Il-ir-i binding polypeptide

ABSTRACT

The present disclosure relates to a class of engineered polypeptides having a binding affinity for interleukin-1 receptor type-I (IL-1R-I) which comprise the binding motif (BM) EX 2 X 3 X 4 X 5 X 6 X 7 EIX 10 X 11 LPNLX 16 RX 18 QYX 21 AFIX 25 X 26 LX 28 D. The present disclosure also relates to the use of such an IL-1R-I binding polypeptide as a therapeutic, prognostic and/or diagnostic agent.

FIELD OF THE INVENTION

The present disclosure relates to a class of engineered polypeptideshaving a binding affinity for interleukin-1 receptor type-I (in thefollowing referred to as IL-1R-1). The present disclosure also relatesto the use of such an IL-1R-I binding polypeptide as a therapeutic,prognostic and/or diagnostic agent.

Background

The central role of the IL-1 system in the initiation and maintenance ofinflammatory response, as well as its role in innate and adaptivebranches of the immune system, makes it an attractive target forpharmaceutical intervention (Dinarello, 2009, Annu Rev Immunol,27:519-50; Sims and Smith, 2010, Nat Rev Immunol.10(2):89-102). Sincethe IL-1 system in many cases plays the role of initiator and mastercytokine in inflammatory response, many aspects of the progression of apathological immune response can be modulated by such intervention. Thebiological activity of IL-1p can be pharmacologically inhibited byantibodies that prevent, or modulate its binding to the type IL-1receptor IL-1R-1.

The biological activity of the IL-1 system can also be modulated byantagonizing the IL-1R. The recombinant form of endogenous IL-1 Radenoted anakinra (Kineret®) is described in e.g. U.S. Pat. No.6,599,873, EP 0343684 and EP 0541920. Anakinra has shown clinicalefficacy in rheumatoid arthritis, cryopyrin-associated periodicsyndromes (CAPS; Koné-Paut and Galeotti, 2014, Expert Rev Clin Immunol,10(1):7-18), as well as in a large number of other inflammatory andautoimmune conditions (Dinarello et al, 2011, Blood, 117(14):3720-32;Dinarello et al, 2012, Nat Rev Drug Discov, 11(8):633-52)).

The IL-1 receptor can also be antagonized by antibodies that bind toIL-1R-I subunit and prevent ligand-mediated IL-1 receptor activation andsignaling. Several such antibodies are described in WO 2004/022718,which discloses IL-1R-I antagonists such as 15C4. Other examples of IL-1receptor antagonizing antibodies binding to the IL-1R-I subunit aredescribed in WO 2010/052505, which discloses IL-1R-I antagonists such asAntibody 9GL. Yet another example of an IL-1 receptor antagonizingantibody, denoted 2D8 is described in WO 2005/023872.

The unpredictable and chronic nature of inflammatory diseases, as wellas a high unmet medical need, warrants the development of new modes oftreatment. Since tissue penetration rate is negatively associated withthe size of the molecule, relatively large antibody molecules inherentlyhave poor tissue distribution and penetration capacity.

Thus, the use of antibodies, or other large molecule drugs, is notalways optimal for therapy and there is continued need for provision ofagents with a high affinity for the IL-1 receptor.

SUMMARY OF THE INVENTION

It is an object of the present disclosure to provide new IL-1R-I bindingagents, which could for example be used for therapeutic, prognostic anddiagnostic applications.

It is an object of the present disclosure to provide a molecule allowingfor efficient therapy targeting various forms of inflammatory,autoinflammatory and autoimmune diseases.

It is furthermore an object of the present disclosure to provide amolecule suitable for prognostic and diagnostic applications.

These and other objects, which are evident to the skilled person fromthe present disclosure, are met by different aspects of the invention asclaimed in the appended claims and as generally disclosed herein.

Thus, in the first aspect of the disclosure, there is provided anIL-1R-I binding polypeptide, comprising an IL-1R-I binding motif BM,which motif consists of an amino acid sequence selected from:

-   i)

(SEQ ID NO: 1686) EX₂X₃X₄X₅X₆X₇EIX₁₀X₁₁LPNLX₁₆RX₁₈QYX₂₁AFIX₂₅X₂₆LX₂₈Dwherein, independently from each other,

-   -   X₂ is selected from A, D, E, F, H, I, L, Q, S, T and V;    -   X₃ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W        and Y;    -   X₄ is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V,        W and Y;    -   X₅ is selected from A, I and V;    -   X₆ is selected from F, H, I, Q, R, T, V and Y;    -   X₇ is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W and        Y;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S,        T, V, W and Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K, R and S;    -   X₂₁ is selected from Q, T and V;    -   X₂₅ is selected from I, M, R, V and Y;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F, I, L and M,    -   and

-   ii) an amino acid sequence which has at least 96% identity to the    sequence defined in i) provided that X₅ is I or V.

The above definition of a class of sequence related, IL-1R-I bindingpolypeptides is based on a statistical analysis of a number of randompolypeptide variants of a parent scaffold, that were selected for theirinteraction with IL-1R-I in several different selection experiments. Theidentified IL-1R-I binding motif, or “BM”, corresponds to the targetbinding region of the parent scaffold, which region constitutes twoalpha helices within a three-helical bundle protein domain. In theparent scaffold, the varied amino acid residues of the two BM helicesconstitute a binding surface for interaction with the constant Fc partof antibodies. In the present disclosure, the random variation ofbinding surface residues and subsequent selection of variants havereplaced the Fc interaction capacity with a capacity for interactionwith IL-1R-I.

As the skilled person will realize, the function of any polypeptide,such as the IL-1R-I binding capacity of the polypeptide of the presentdisclosure, is dependent on the tertiary structure of the polypeptide.It is therefore possible to make minor changes to the sequence of aminoacids in a polypeptide without affecting the function thereof. Thus, thedisclosure encompasses modified variants of the IL-1R-I bindingpolypeptide, which have retained IL-1R-I binding characteristics.

In this way, also encompassed by the present disclosure is an IL-1R-Ibinding polypeptide comprising an amino acid sequence with 96% orgreater identity to a polypeptide as defined in i). Thus, certainsequence variants of the sequence defined in i) are encompassed by theabove definition under the proviso that within such sequence variants X₅is I or V. For example, it is possible that an amino acid residuebelonging to a certain functional grouping of amino acid residues (e.g.hydrophobic, hydrophilic, polar etc) could be exchanged for anotheramino acid residue from the same functional group.

In some embodiments, such changes may be made in any position of thesequence of the IL-1R-I binding polypeptide as disclosed herein. Inother embodiments, such changes may be made only in the non-variablepositions, also denoted scaffold amino acid residues. In such cases,changes are not allowed in the variable positions, i.e. positionsdenoted with an “X” in sequence i).

The term “% identity”, as used throughout the specification, may forexample be calculated as follows. The query sequence is aligned to thetarget sequence using the CLUSTAL W algorithm (Thompson et al, 1994,Nucleic Acids Research, 22: 4673-4680). A comparison is made over thewindow corresponding to the shortest of the aligned sequences. Theshortest of the aligned sequences may in some instances be the targetsequence. In other instances, the query sequence may constitute theshortest of the aligned sequences. The amino acid residues at eachposition are compared and the percentage of positions in the querysequence that have identical correspondences in the target sequence isreported as % identity. As used herein “X_(a)” and “X_(m)” are used toindicate amino acids in positions n and m in the sequence i) as definedabove, wherein n and m are integers which indicate the position of anamino acid within said sequence as counted from the N-terminal end ofsaid sequence. For example, X₃ and X₇ indicate the amino acid inposition three and seven, respectively, from the N-terminal end ofsequence i).

In embodiments according to the first aspect, there are providedpolypeptides wherein X_(n) in sequence i) is independently selected froma group of possible residues according to Table 1. The skilled personwill appreciate that X_(n) may be selected from any one of the listedgroups of possible residues and that this selection is independent fromthe selection of amino acids in X_(m), wherein n≠m. Thus, any of thelisted possible residues in position X_(n) in Table 1 may beindependently combined with any of the listed possible residues in anyother variable position in Table 1.

The skilled person will appreciate that Table 1 is to be read asfollows: In one embodiment according to the first aspect, there isprovided a polypeptide wherein amino acid residue “X_(n)” in sequence i)is selected from “Possible residues”. Thus, Table 1 discloses severalspecific and individualized embodiments of the first aspect of thepresent disclosure. For example, in one embodiment according to thefirst aspect, there is provided a polypeptide wherein X₄ in sequence i)is selected from A, D, E, I, K, O, S and V, and in another embodimentaccording to the first aspect, there is provided a polypeptide whereinX₄ in sequence i) is selected from A, E, I, K, Q and V. For avoidance ofdoubt, the listed embodiments may be freely combined in yet otherembodiments. For example, one such combined embodiment is a polypeptidein which X₄ is selected from A, E, I, K, Q and V, while X₆ is selectedfrom H, Q, T and Y, and X₁ is selected from H, R and Y.

TABLE 1 Embodiments of the first aspect of the present disclosureResidue X_(n) Possible residues X₂ A, E, F, H, I, L, Q, S, T, V X₂ A, F,H, I, L, Q, S, T, V X₂ A, H, I, L, Q, S, T, V X₂ A, H, I, L, Q, T, V X₂A, H, I, L, T, V X₂ A, F, I, L, T, V X₂ A, I, L, T, V X₂ A, I, T, V X₂A, I, L, T X₂ A, L, T, V X₂ A, I, L, V X₂ I, L, T, V X₂ A, I, T X₂ A, I,V X₂ A, T, V X₂ I, T, V X₂ I, L, T X₂ I, L, V X₂ L, T, V X₂ I, V X₂ I, LX₂ I, T X₂ I, V X₂ L, T X₂ L, V X₂ T, V X₂ A, I X₂ A, T X₂ A, V X₂ L X₂T X₂ V X₂ I X₂ A X₃ A, D, E, F, H, I, K, L, Q, R, S, T, V, W, Y X₃ A, D,E, F, H, I, K, L, N, Q, R, S, T, V, Y X₃ A, D, E, H, I, K, L, N, Q, R,S, T, V, Y X₃ A, D, E, F, H, I, K, Q, R, S, T, V, Y X₃ A, D, E, F, H, I,K, Q, R, V, Y X₃ D, E, H, I, K, L, Q, T, V, Y X₃ D, E, H, I, K, Q, T, V,Y X₃ D, E, H, I, K, Q, R, S, V, Y X₃ D, E, H, I, K, Q, S, V, Y X₃ D, E,H, I, K, Q, R, V, Y X₃ D, E, I, K, Q, R, V, Y X₃ D, E, H, I, K, Q, V, YX₃ D, E, H, I, Q, V, Y X₃ D, E, I, Q, R, V, Y X₃ D, E, I, Q, V, Y X₃ D,E, H, Q, Y X₃ D, E, Q, V, Y X₃ D, E, V, Y X₃ D, E, R, V X₃ D, E, K, V X₃E, I, V, Y X₃ E, V, Y X₃ D, E, V X₃ D, E, Y X₃ E, V X₃ D, E X₃ E, Y X₃E, I X₃ E, K X₃ E X₄ A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W, Y X₄A, D, E, F, H, I, K, L, N, Q, R, S, T, V, Y X₄ A, D, E, F, H, I, K, L,Q, R, S, T, V, W, Y X₄ A, D, E, F, H, I, K, Q, R, S, T, V, W, Y X₄ A, D,E, F, H, K, L, Q, R, S, T, V, W, Y X₄ A, D, E, H, I, K, L, Q, R, S, T,V, W, Y X₄ A, D, E, H, I, K, L, N, Q, R, S, T, V, Y X₄ A, D, E, I, K, Q,R, S, T, V X₄ A, D, E, I, K, Q, T, V X₄ A, D, E, H, I, K, N, Q, R, S, T,V, Y X₄ A, D, E, H, I, K, Q, R, S, T, V, W, Y X₄ A, D, E, I, K, Q, R, S,T, V X₄ A, E, I, K, Q, R, T, V, Y X₄ A, D, E, H, I, K, Q, R, S, T, V X₄A, D, E, H, I, K, R, S, V X₄ A, D, E, H, K Q V, W X₄ A, D, E, H, I, R,S, T, V X₄ A, D, E, I, K, Q, S, V X₄ A, D, E, I, Q, R, V X₄ A, D, E, K,Q, S, T, V X₄ A, D, E, I, Q, V X₄ A, D, E, K, Q, V X₄ A, E, I, K, Q, VX₄ A, E, H, K, Q, V X₄ A, D, E, I, V X₄ A, D, E, I, R X₄ D, E R, T, V X₄A, D, E, K, Q X₄ A, D, E, Q, V X₄ A, D, E, K, V X₄ A, E, Q, V X₄ A, E,I, R X₄ A, D, E, I X₄ A, D, E, Q X₄ A, D, E, V X₄ A, D, E, K X₄ A, E, K,V X₄ A, D, E X₄ A, E, V X₄ A, D X₄ A, E X₄ D, E X₄ E, V X₄ E X₄ D X₄ AX₄ V X₅ A, V X₅ I, V X₅ A, I X₅ A X₅ I X₅ V X₆ F, H, Q, R, V, Y X₆ H, Q,T, Y X₆ F, H, Q, Y X₆ A, H, Q, W, Y X₆ H, Q, Y X₆ Q, Y X₆ Y X₆ Q X₇ A,D, E, F, H, I, L, M, Q, S, V, W, Y X₇ A, D, E, F, H, L, M, Q, R, W, Y X₇A, D, E, F, H, I, L, M, Q, W, Y X₇ D, E, F, H, L, M, Q, W, Y X₇ D, F, H,L, M, Q, W, Y X₇ F, H, I, L, M, Q, V, W, Y X₇ F, H, I, L, M, Q, V, Y X₇D, F, H, L, M, Q, W, Y X₇ F, H, I, L, M, Q, Y X₇ F, L, M, Q, W, Y X₇ F,M, Q, W, Y X₇ F, L, M, W, Y X₇ F, I, L, M, Y X₇ F, H, M, W, Y X₇ F, L,M, Y X₇ F, M, W, Y X₇ F, L, M, W X₇ D, F, H, M X₇ D, F, M X₇ F, M, Y X₇M, W, Y X₇ F, M, W X₇ F, L, M X₇ L, M, Y X₇ F, L, Y X₇ F, M X₇ L, M X₇M, Y X₇ F, L X₇ L, Y X₇ F, Y X₇ F, W X₇ M, W X₇ L X₇ Y X₇ F X₇ M X₇ WX₁₀ F X₁₀ Y X₁₁ A, D, E, F, G, H, I, K, L, N, Q, R, S, T, V, Y X₁₁ A, D,E, F, G, H, K, L, N, Q, R, S, T, V, W, Y X₁₁ A, D, E, F, G, H, I, K, N,Q, R, S, T, V, Y X₁₁ A, D, E, F, G, H, K, L, N, Q, R, S, T, V, Y X₁₁ A,D, E, F, G, H, K, L, Q, R, S, T, V, W, Y X₁₁ A, D, E, F, G, H, K, L, N,Q, R, S, Y X₁₁ A, D, E, F, G, H, K, L, Q, R, S, T, V, Y X₁₁ A, D, E, F,G, H, K, L, Q, R, S, T Y X₁₁ A, D, E, F, G, H, K, L, N, Q, R, S, T, YX₁₁ A, D, E, F, G, H, K, N, Q, R, S, T, V, Y X₁₁ A, D, E, F, G, H, K, N,Q, R, S, V, Y X₁₁ A, D, E, F, G, H, K, Q, R, S, V, W, Y X₁₁ A, D, E, F,G, H, K, Q, R, S, V, Y X₁₁ A, D, E, G, H, K, N, Q, R, S, Y X₁₁ A, D, E,F, G, H, I, L, M, Q, S, T, V, W, Y X₁₁ A, D, E, G, K, N, Q, R, S, T, YX₁₁ A, D, E, F, G, H, Q, R, S, Y X₁₁ A, D, E, G, H, Q, S, Y X₁₁ A, D, E,F, G, H, K, Q, R, S, Y X₁₁ A, D, G, H, K, Q, R, S, Y X₁₁ A, E, G, H, K,N, Q, R, S, Y X₁₁ A, E, G, H, K, N, R, S, Y X₁₁ A, E, G, H, K, N, S, YX₁₁ A, E, G, H, N, R, S, Y X₁₁ A, D, G, H, K, Q, R, S X₁₁ A, D, G, H, Q,R, S, Y X₁₁ A, E, G, H, R, S, Y X₁₁ A, D, G, H, Q, R, S X₁₁ A, G, H, Q,R, S, Y X₁₁ A, G, H, Q, R, S X₁₁ A, E, G, H, S, Y X₁₁ A, E, G, N, S, YX₁₁ A, H, R, S, Y X₁₁ A, D, G, H, S, Y X₁₁ A, D, G, H, R, Y X₁₁ A, E, N,R, Y X₁₁ A, E, G, H, Y X₁₁ A, G, H, S, Y X₁₁ A, E, G, S, Y X₁₁ A, E, G,H, S X₁₁ A, E, H, R, Y X₁₁ A, G, H, R, Y X₁₁ A, H, R, S, Y X₁₁ A, E, G,N, S X₁₁ A, H, N, R, Y X₁₁ A, D, G, H X₁₁ A, G, S, Y X₁₁ A, G, H, Y X₁₁A, G, H, S X₁₁ A, E, G, Y X₁₁ A, E, G, S X₁₁ A, E, G, H X₁₁ A, H, R, YX₁₁ G, H, R, Y X₁₁ A, G, R, Y X₁₁ A, G, H, Y X₁₁ A, G, H, R X₁₁ A, E, G,N X₁₁ A, E, G, S X₁₁ A, E, G X₁₁ A, G, H X₁₁ A, G, S X₁₁ A, G, Y X₁₁ A,H, R X₁₁ A, R, Y X₁₁ A, H, Y X₁₁ H, R, Y X₁₁ A, G X₁₁ A, H X₁₁ A, R X₁₁A, Y X₁₁ H, R X₁₁ H, Y X₁₁ R, Y X₁₁ A, E X₁₁ A, X₁₁ G X₁₁ R X₁₁ Y X₁₁ HX₁₁ E X₁₆ N X₁₆ T X₁₈ K, R X₁₈ R X₁₈ K X₂₁ T, V X₂₁ T X₂₁ V X₂₅ I, M, RX₂₅ I, R X₂₅ R X₂₅ I X₂₆ K X₂₆ S X₂₈ F, I, L X₂₈ F, L X₂₈ F X₂₈ L

In one embodiment of the present disclosure there is provided an IL-1R-Ibinding polypeptide, wherein in i)

-   -   X₂ is selected from A, E, F, H, I, L, Q, S, T and V;    -   X₃ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W        and Y;    -   X₄ is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V,        W and Y;    -   X₅ is selected from A, I and V;    -   X₆ is selected from F, H, Q, R, V and Y;    -   X₇ is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W and        Y;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W        and Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K and R;    -   X₂₁ is selected from T and V;    -   X₂₅ is selected from I and R;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F and L.

In one embodiment of the present disclosure there is provided an IL-1R-Ibinding polypeptide, wherein in i)

-   -   X₂ is selected from A, E, F, H, I, L, Q, S, T and V;    -   X₃ is selected from A, D, E, F, H, I, K, L, Q, R, S, T, V, W and        Y;    -   X₄ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W        and Y;    -   X₅ is selected from A, I and V;    -   X₆ is selected from H, Q, R, and Y;    -   X₇ is selected from A, D, E, F, G, H, I, L, M, Q, S, V, W and Y;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, I, K, L, N, Q, R, S, T,        V, W and Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K and R;    -   X₂₁ is selected from T and V;    -   X₂₅ is selected from I and R;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F and L.

In one embodiment of the present disclosure there is provided an IL-1R-Ibinding polypeptide, wherein in i)

-   -   X₂ is selected from A, I, L, T and V;    -   X₃ is selected from E, I, V and Y;    -   X₄ is selected from A, D, E, F, H, I, K, L, Q, R, S, T, V, W and        Y;    -   X₅ is selected from A, I and V;    -   X₆ is selected from Q and Y;    -   X₇ is selected from F, H, I, L, M, Q, V, W and Y;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V, W        and Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K and R;    -   X₂₁ is selected from T and V;    -   X₂₅ is selected from I and R;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F and L.

In another embodiment of the present disclosure there is provided anIL-1R-I binding polypeptide, wherein said IL-1R-I binding motif consistsof an amino acid sequence selected from a sequence wherein in i)

-   -   X₂ is selected from A, I, L, T and V;    -   X₃ is selected from E and Y;    -   X₄ is selected from A, E, I, K, Q, R, T, V and Y;    -   X₅ is selected from I and V;    -   X₆ is selected from Q and Y;    -   X₇ is selected from F and M;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V and        Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K and R;    -   X₂₁ is selected from T and V;    -   X₂₅ is selected from I and R;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F and L,        and an amino acid sequence which has at least 93% identity to        the sequence defined above.

In another embodiment of the present disclosure there is provided anIL-1R-I binding polypeptide, wherein in i)

-   -   X₂ is selected from A, I, L, T and V;    -   X₃ is selected from E and Y;    -   X₄ is selected from A, D, E, F, H, K, L, Q, R, S, T, V, W and Y;    -   X₅ is selected from A, I and V;    -   X₆ is selected from Q and Y;    -   X₇ is selected from F, H, I, L, M, Q, V, W and Y, such as F, H,        I, L, Q, V, W and Y;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, K, Q, R, S, V, W and Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K and R;    -   X₂₁ is T;    -   X₂₅ is selected from I and R;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F and L.

In a more specific embodiment defining a sub-class of IL-1R-I bindingpolypeptides, sequence i) fulfills at least five of the ten conditionsI-X:

-   -   I. X₃ is E;    -   II. X₅ is selected from I and V;    -   Ill. X₆ is selected from Q and Y;    -   IV. X₇ is selected from F and M;    -   V. X₁₀ is selected from F and Y;    -   VI. X₁₈ is selected from K and R;    -   VII. X₂₁ is T;    -   VIII. X₂₅ is R;    -   IX. X₂₆ is selected from K and S; and    -   X. X₂₈ is selected from F and L.

In some examples of an IL-1R-I binding polypeptide according to thefirst aspect, sequence i) fulfils at least six of the ten conditionsI-X. More specifically, sequence i) may fulfil at least seven of the tenconditions I-X, such as at least eight of the ten conditions I-X, suchas at least nine of the ten conditions I-X, such as all of the tenconditions I-X.

In some embodiments of an IL-1R-I binding polypeptide according to thefirst aspect, there is provided an IL-1R-I binding polypeptide, whereinX₂X₃X₆ is VEQ or VEY. In some embodiments there is provided an IL-1R-Ibinding polypeptide, wherein X₂X₃X₆ is IEQ or VEQ. In some embodimentsX₆X₁₀ is selected from the group consisting of QF, QY, YF and YY. Insome embodiments X₆X₁₀ is QF. In some embodiments X₁₀X₁₈ is selectedfrom the group consisting of FK, FR, YK and YR. In some embodimentsX₁₀X₁₈ is FK or FR. In some embodiments X₁₈X₂₅X₂₈ is KRL or RRL. In someembodiments X₅X₇ is AM, IM or VM. In some embodiments X₅X₇ is IM or VM.

In one embodiment, X₅ is selected from I and V. As demonstrated in theappended Example 4, the unintentional alteration in position X₅ of theBM resulted in the finding of an IL-1R-I binding polypeptide (Z18557,SEQ ID NO:1205) displaying high affinity for IL-1R-I (see Table 11). Theexchange of alanine in this position to valine or isoleucine allows forgeneration of IL-1R-I binding polypeptides with high affinity forIL-1R-I.

As described in detail in the experimental section to follow, theselection of IL-1R-I binding polypeptide variants has led to theidentification of a number of individual IL-1R-I binding motif (BM)sequences. These sequences constitute individual embodiments of sequencei) according to this aspect. The sequences of individual IL-1R-I bindingmotifs correspond to amino acid positions 8-36 of the amino acidsequences listed as SEQ ID NOs 1-1632 and 1679 presented in FIG. 1.Thus, in one embodiment, of the IL-1R-I binding polypeptide according tothis aspect, sequence i) corresponds to the sequence from position 8 toposition 36 in a sequence selected from the group consisting of SEQ IDNO:1-1632 and 1679, such as the group consisting SEQ ID NO:20-1632, and1679. In one embodiment sequence i) corresponds to the sequence fromposition 8 to position 36 in a sequence selected from the groupconsisting of SEQ ID NO:1206-1632 and 1679, such as the group consistingof SEQ ID NO:1210-1632 and 1679.

In one embodiment sequence i) corresponds to the sequence from position8 to position 36 in a sequence selected from the group consisting of SEQID NO:1205 and 1250-1632.

In one embodiment sequence i) corresponds to the sequence from position8 to position 36 in a sequence selected from the group consisting of SEQID NO:1206-1252 and 1679.

In another embodiment sequence i) corresponds to the sequence fromposition 8 to position 36 in a sequence selected from the groupconsisting of SEQ ID NO:1253-1632. In another embodiment sequence i)corresponds to the sequence from position 8 to position 36 in a sequenceselected from the group consisting of SEQ ID NO:1253-1441 or the groupconsisting of SEQ ID NO:1442-1632.

In one embodiment, sequence i) corresponds to the sequence from position8 to position 36 in a sequence selected from the group consisting of SEQID NO:158, 227, 268, 294, 325, 1176, 1205, 1251, 1252, 1270, 1284, 1285,1298, 1307, 1308, 1311, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343,1353, 1361, 1362, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421, 1422,1423, 1435, 1471, 1472, 1571, 1594 and 1662, such as from the groupconsisting of SEQ ID NO:268, 1205, 1252, 1270, 1284, 1285, 1298, 1307,1308, 1324, 1328, 1330, 1331, 1334, 1339, 1340, 1343, 1361, 1364, 1367,1368, 1375, 1393, 1415, 1420, 1421, 1422, 1423 and 1571, such as fromthe group consisting of SEQ ID NO:1252, 1285, 1298, 1308, 1324, 1328,1330, 1331, 1340, 1361, 1393, 1415, 1420 and 1421.

In one particular embodiment sequence i) corresponds to the sequencefrom position 8 to position 36 in a sequence selected from the groupconsisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421,1435, 1594 and 1679. In one embodiment sequence i) corresponds to thesequence from position 8 to position 36 in a sequence selected from thegroup consisting of SEQ ID NO:1252, 1328, 1435 and 1679, such as thegroup consisting of SEQ ID NO:1252, 1328 and 1679; the group consistingof SEQ ID NO:1252, 1435, 1679; the group consisting of SEQ ID NO:1252and 1679; or the group consisting of SEQ ID NO:1328 and 1435. In oneembodiment, sequence i) corresponds to the sequence from position 8 toposition 36 in sequence selected from the group consisting of SEQ IDNO:1252, 1328 and 1435.

FIG. 1 thus exemplifies polypeptide variants whose IL-1R-I bindingcapacity has been verified experimentally (at least by virtue of beingisolated in an affinity based assay), which variants have led to theidentification of a number of individual IL-1R-I binding motif (BM)sequences which constitute individual embodiments of BM which exhibit93%, where applicable, or 96% identity with sequence i).

In some embodiments of the present disclosure, the BM as defined above“forms part of” a three-helix bundle protein domain. This is understoodto mean that the sequence of the BM is “inserted” into or “grafted” ontothe sequence of the original three-helix bundle domain, such that the BMreplaces a similar structural motif in the original domain. For example,without wishing to be bound by theory, the BM is thought to constitutetwo of the three helices of a three-helix bundle, and can thereforereplace such a two-helix motif within any three-helix bundle. As theskilled person will realize, the replacement of two helices of thethree-helix bundle domain by the two BM helices has to be performed soas not to affect the basic structure of the polypeptide. That is, theoverall folding of the Ca backbone of the polypeptide according to thisembodiment of the invention is substantially the same as that of thethree-helix bundle protein domain of which it forms a part, e.g. havingthe same elements of secondary structure in the same order etc. Thus, aBM according to the present disclosure “forms part” of a three-helixbundle domain if the polypeptide according to this embodiment has thesame fold as the original domain, implying that the basic structuralproperties are shared, those properties e.g. resulting in similar CDspectra. The skilled person is aware of other parameters that arerelevant.

In particular embodiments, the IL-1R-I binding motif (BM) thus formspart of a three-helix bundle protein domain. For example, the BM mayessentially constitute two alpha helices with an interconnecting loop,within said three-helix bundle protein domain. In particularembodiments, said three-helix bundle protein domain is selected fromdomains of bacterial receptor proteins. Non-limiting examples of suchdomains are the five different three-helical domains of Protein A fromStaphylococcus aureus, such as domain B, and derivatives thereof. Insome embodiments, the three-helical bundle protein domain is a variantof protein Z, which is derived from domain B of staphylococcal ProteinA.

In some embodiments where the IL-1R-I binding polypeptide as disclosedherein forms part of a three-helix bundle protein domain, the IL-1R-Ibinding polypeptide may comprise a binding module (BMod), the amino acidsequence of which is selected from:

(SEQ ID NO: 1687) K-[BM]-DPSQSX_(a)X_(b)LLX_(c)EAKKLX_(d)X_(e)X_(f)Q;wherein[BM] is an IL-1R-I binding motif as defined herein;X_(a) is selected from A and S;X_(b) is selected from N and E;X_(c) is selected from A, S and C;X_(d) is selected from E, N and S;X_(e) is selected from D, E and S;X_(f) is selected from A and S; and

-   -   iv) an amino acid sequence which has at least 91% identity to a        sequence defined by iii).

In some embodiments, said polypeptides may beneficially exhibit highstructural stability, such as resistance to chemical modifications, tochanges in physical conditions and to proteolysis, during production andstorage, as well as in vivo.

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences, which do not largely affect the tertiarystructure and the function of the polypeptides, are also within thescope of the present disclosure. Thus, in some embodiments, sequence iv)has at least at least 93%, such as at least 95%, such as at least 97%identity to a sequence defined by iii). In particular embodiments, suchminor changes as exemplified above may be present only in the flankingsequence, i.e. the parts of the sequence flanking the BM in iii) above.In other embodiments, minor changes can be present in the BM and/or inthe flanking sequences.

In one embodiment, X_(a) in sequence iii) is A.

In one embodiment, X_(a) in sequence iii) is S.

In one embodiment, X_(b) in sequence iii) is N.

In one embodiment, X_(b) in sequence iii) is E.

In one embodiment, X_(c) in sequence iii) is A.

In one embodiment, X_(c) in sequence iii) is S.

In one embodiment, X_(c) in sequence iii) is C.

In one embodiment, X_(d) in sequence iii) is E.

In one embodiment, X_(d) in sequence iii) is N.

In one embodiment, X_(d) in sequence iii) is S.

In one embodiment, X_(e) in sequence iii) is D.

In one embodiment, X_(e) in sequence iii) is E.

In one embodiment, X_(e) in sequence iii) is S.

In one embodiment, X_(d)X_(e) in sequence iii) is selected from EE, ES,SE and SS.

In one embodiment, X_(d)X_(e) in sequence iii) is ES.

In one embodiment, X_(d)X_(e) in sequence iii) is SE.

In one embodiment, X_(f) in sequence iii) is A.

In one embodiment, X_(f) in sequence iii) is S.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is Aand X_(f) is A.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is Cand X_(f) is A.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is Sand X_(f) is S.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is Cand X_(f) is S.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is A;X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is C;X_(d)X_(e) is ND and X_(f) is A.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is S;X_(d)X_(e) is ND and X_(f) is S.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is C;X_(d)X_(e) is ND and X_(f) is S.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is A;X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is C;X_(d)X_(e) is SE and X_(f) is A.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is S;X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is C;X_(d)X_(e) is SE and X_(f) is S.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is A;X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii), X_(a) is A; X_(b) is N; X_(c) is C;X_(d)X_(e) is ES and X_(f) is A.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is S;X_(d)X_(e) is ES and X_(f) is S.

In one embodiment, in sequence iii), X_(a) is S; X_(b) is E; X_(c) is C;X_(d)X_(e) is ES and X_(f) is S.

In one embodiment, sequence iii) or iv) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1-1638 and 1667-1670.

In yet a further embodiment, sequence iii) corresponds to the sequencefrom position 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1-1638, 1667-1668 and 1670-1679; such as thegroup consisting of SEQ ID NO:20-1638 and 1670-1679. In one embodimentsequence iii) corresponds to the sequence from position 7 to position 55in a sequence selected from the group consisting of SEQ ID NO:1206-1638and 1670-1679, such as the group consisting of SEQ ID NO:1206-1632 and1670-1679, such as the group consisting of SEQ ID NO:1210-1638 and1670-1679, such as the group consisting of SEQ ID NO:1210-1632 and1670-1679.

In one embodiment sequence iii) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1205, 1250-1632 and 1670-1679.

In one embodiment sequence iii) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1206-1252, 1672 and 1679.

In another embodiment sequence iii) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1253-1632, 1670-1671 and 1673-1678. In anotherembodiment sequence iii) corresponds to the sequence from position 7 toposition 55 in a sequence selected from the group consisting of SEQ IDNO:1253-1441, 1670-1671 and 1674-1678 or the group consisting of SEQ IDNO:1442-1632 and 1673.

In one embodiment, sequence iii) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:158, 227, 268, 294, 325, 1176, 1205, 1251, 1252,1270, 1284, 1285, 1298, 1307, 1308, 1311, 1324, 1328, 1330, 1331, 1334,1339, 1340, 1343, 1353, 1361, 1362, 1364, 1367, 1368, 1375, 1393, 1415,1420, 1421, 1422, 1423, 1435, 1471, 1472, 1571, 1594, 1662 and1670-1679, such as from the group consisting of SEQ ID NO:268, 1205,1252, 1270, 1284, 1285, 1298, 1307, 1308, 1324, 1328, 1330, 1331, 1334,1339, 1340, 1343, 1361, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421,1422, 1423, 1571, 1670-1672, 1674, and 1676-1679, such as from the groupconsisting of SEQ ID NO:1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331,1340, 1361, 1393, 1415, 1420, 1421, 1670-1672, 1674, 1676-1677 and 1679.

In one particular embodiment sequence iii) corresponds to the sequencefrom position 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331, 1415, 1421,1435, 1594 and 1670-1679. In one embodiment sequence iii) corresponds tothe sequence from position 7 to position 55 in a sequence selected fromthe group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676 and1679 such as the group consisting of SEQ ID NO:1252, 1328 and 1679; thegroup consisting of SEQ ID NO:1252, 1435 and 1679; or the groupconsisting of SEQ ID NO:1328 and 1435. In one embodiment, sequence i)corresponds to the sequence from position 7 to position 55 in sequenceselected from the group consisting of SEQ ID NO:1252, 1328 and 1435.

In one embodiment, sequence iii) corresponds to the sequence fromposition 7 to position 55 in a sequence selected from the groupconsisting of SEQ ID NO:1672, 1675-1676 and 1679, such as the groupconsisting of SEQ ID NO:1672 and 1675-1676.

Also, in a further embodiment, there is provided an IL-1R-I bindingpolypeptide, which comprises an amino acid sequence selected from:

v) (SEQ ID NO: 1688) YA-[BMod]-AP;wherein [BMod] is an IL-1R-I binding module as defined herein; and

vi) an amino acid sequence which has at least 90% identity to a sequencedefined in v).

Alternatively, there is provided an IL-1R-I binding polypeptide, whichcomprises an amino acid sequence selected from:

vii) (SEQ ID NO: 1689) FN-[BMod]-AP;wherein [BMod] is an IL-1R-I binding module as defined herein; and

viii) an amino acid sequence which has at least 90% identity to asequence defined in vii).

In one embodiment there is provided an IL-1R-I binding polypeptide,which comprises an amino acid selected from the group consisting of

ix) (SEQ ID NO: 1690) FNK-[BM]-DPSQS ANLLX_(c) EAKKL NDAQA P;wherein [BM] is an IL-1R-I binding motif as defined above and X_(c) isselected from A and C; and

x) an amino acid sequence which has at least 90% identity to a sequencedefined in ix).

In another embodiment, there is provided an IL-1R-I binding polypeptide,which comprises an amino acid selected from the group consisting of

xi) (SEQ ID NO: 1691) FAK-[BM]-DPSQS SELLX_(c) EAKKL SESQA P;wherein [BM] is an IL-1R-I binding motif as defined above and X_(c) isselected from A, S and C; and

xii) an amino acid sequence which has at least 90% identity to asequence defined in xi).

In another embodiment, there is provided an IL-1R-I binding polypeptide,which comprises an amino acid selected from the group consisting of

xiii) (SEQ ID NO: 1692) FAK-[BM]-DPSQS SELLX_(c) EAKKL NDSQA P;wherein [BM] is an IL-1R-I binding motif as defined above and X_(c) isselected from A, S and C;

xiv) an amino acid sequence which has at least 90% identity to asequence defined in xiii).

In yet another embodiment, there is provided an IL-1R-I bindingpolypeptide, which comprises an amino acid selected from the groupconsisting of

xv) (SEQ ID NO: 1693) YAK-[BM]-DPSQS SELLX_(c) EAKKL X_(d)X_(e)SQA P;wherein [BM] is an IL-1R-I binding motif as defined above and X_(c) isselected from A, S and C, X_(d) is selected from E, N and S, and X_(e)is selected from D, E and S;

xvi) and an amino acid sequence which has at least 90% identity to asequence defined in xv).

In yet another embodiment, there is provided an IL-1R-I bindingpolypeptide, which comprises an amino acid selected from the groupconsisting of

xvii) (SEQ ID NO: 1694) KYAK-[BM]-DPSQS SELLX_(c) EAKKL X_(d)X_(e)SQA P;wherein [BM] is an IL-1R-I binding motif as defined above and X_(c) isselected from A, S and C, X_(d) is selected from E, N and S, and X_(e)is selected from D, E and S;

xviii) and an amino acid sequence which has at least 90% identity to asequence defined in xvii).

As discussed above, polypeptides comprising minor changes as compared tothe above amino acid sequences, which do not largely affect the tertiarystructure and the function of the polypeptide, also fall within thescope of the present disclosure. Thus, in some embodiments, sequencevi), viii), x), xii), xiv), xvi) or xviii) may for example be at least90%, such as at least 92%, such as at least 94%, such as at least 96%,such as at least 98% identical to a sequence defined by v), vii), ix),xi), xiii), xv) and xvii), respectively. In particular embodiments, suchminor changes as exemplified above can be present only in flankingsequences, i.e. the parts of the sequences flanking the BM in v), vii),ix), xi), xiii), xv) and xviii). In other embodiments, minor changes canbe present in the BM and/or in the flanking sequences.

In some embodiments, the IL-1R-I binding motif may form part of apolypeptide comprising an amino acid sequence selected from:

SEQ ID NO: 1695 ADNNFNK-[BM]DPSQSANLLSEAKKLNESQAPK; SEQ ID NO: 1696ADNKFNK-[BM]DPSQSANLLAEAKKLNDAQAPK; SEQ ID NO: 1697ADNKFNK-[BM]DPSVSKEILAEAKKLNDAQAPK; SEQ ID NO: 1698ADAQQNNFNK-[BM]DPSQSTNVLGEAKKLNESQAPK; SEQ ID NO: 1699AQHDE-[BM]DPSQSANVLGEAQKLNDSQAPK; SEQ ID NO: 1700VDNKFNK-[BM]DPSQSANLLAEAKKLNDAQAPK; SEQ ID NO: 1701AEAKYAK-[BM]DPSESSELLSEAKKLNKSQAPK; SEQ ID NO: 1702VDAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1703VDAKYAK-[BM]DPSQSSELLAEAKKLNDSQAPK; SEQ ID NO: 1704AEAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1705AEAKYAK-[BM]DPSQSSELLSEAKKLNDSQAP; SEQ ID NO: 1706AEAKFAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1707AEAKFAK-[BM]DPSQSSELLSEAKKLNDSQAP; SEQ ID NO: 1708AEAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1709AEAKYAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1710AEAKYAK-[BM]DPSQSSELLSEAKKLSESQAP; SEQ ID NO: 1711AEAKFAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1712AEAKFAK-[BM]DPSQSSELLSEAKKLSESQAP; SEQ ID NO: 1713AEAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; SEQ ID NO: 1714AEAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; SEQ ID NO: 1715AEAKYAK-[BM]DPSQSSELLSEAKKLESSQAP; SEQ ID NO: 1716AEAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; SEQ ID NO: 1717AEAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; SEQ ID NO: 1718AEAKYAK-[BM]DPSQSSELLSEAKKLSDSQAP; SEQ ID NO: 1719AEAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; SEQ ID NO: 1720AEAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; SEQ ID NO: 1721VDAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1722VDAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1723VDAKYAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1724VDAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; SEQ ID NO: 1725VDAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; SEQ ID NO: 1726VDAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; SEQ ID NO: 1727VDAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; SEQ ID NO: 1728VDAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; SEQ ID NO: 1729VDAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; SEQ ID NO: 1730VDAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK; SEQ ID NO: 1731AEAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK, and SEQ ID NO: 1732ADAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK,wherein [BM] is an IL-1R-I binding motif as defined herein.

In one embodiment, said IL-1R-I binding polypeptide comprises an aminoacid sequence which has at least 89% identity to any one of thesequences defined above.

In one embodiment, the IL-1R-I binding polypeptide comprises an aminoacid sequence selected from:

xix) (SEQ ID NO: 1721) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPKwherein [BM] is an IL-1R-I binding motif as defined herein; and

xx) an amino acid sequence which has at least 89% identity to thesequence defined in xix).

In one embodiment, the IL-1R-I binding polypeptide comprises an aminoacid sequence selected from:

xxi) (SEQ ID NO: 1709) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPKwherein [BM] is an IL-1R-I binding motif as defined herein; and

xxii) an amino acid sequence which has at least 89% identity to thesequence defined in xxi).

In one particular embodiment, said IL-1R-I binding polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:1667-1668 and 1670-1679.

In one embodiment, the IL-1R-I binding polypeptide comprises an aminoacid sequence selected from:

xxiii) AEAKFAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQ ID NO:1711) wherein [BM]is an IL-1R-I binding motif as defined herein; and

xxiv) an amino acid sequence which has at least 89% identity to thesequence defined in xxiii).

Again, polypeptides comprising minor changes as compared to the aboveamino acid sequences, which do not largely affect the tertiary structureand the function of the polypeptide, also fall within the scope of thepresent disclosure. Thus, in some embodiments, sequence xx), xxii) orxxiv) may for example be at least 89%, such as at least 91%, such as atleast 93%, such as at least 94%, such as at least 96%, such as at least98% identical to a sequence defined by xix), xxi) or xxiii)respectively. Once again, in particular embodiments such minor changesas exemplified above can be present only in the sequences flanking theBM as set out in xix), xxi) or xxiii). In other embodiments, minorchanges can be present in the BM and/or in the flanking sequences.

Sequence xix)-xxiv) in such IL-1R-I binding polypeptide may be selectedfrom the sequence from position 1 to position 58 in a sequence selectedfrom the group consisting of SEQ ID NO:1-1638, 1667-1668 and 1670-1679as presented in FIG. 1.

In one embodiment of the IL-1R-I binding polypeptide according to thisaspect, sequence xix), xxi) or xxiii) corresponds to the sequence fromposition 1 to position 58 in a sequence selected from the groupconsisting of SEQ ID NO:1-1632, 1667-1668 and 1670-1679; such as thegroup consisting of SEQ ID NO:20-1638, 1667-1668 and 1670-1679. In oneembodiment sequence xix), xxi) or xxiii) corresponds to the sequencefrom position 1 to position 58 in a sequence selected from the groupconsisting of SEQ ID NO:1206-1632, 1667-1668 and 1670-1679, such as thegroup consisting of SEQ ID NO:1210-1632, 1667-1668 and 1670-1679.

In one embodiment sequence xvii), xix) or xxi) corresponds to thesequence from position 1 to position 58 in a sequence selected from thegroup consisting of SEQ ID NO:1205, 1250-1632 and 1670-1679.

In one embodiment sequence xix), xxi) or xxiii) corresponds to thesequence from position 1 to position 58 in a sequence selected from thegroup consisting of SEQ ID NO:1206-1252, 1672 and 1679.

In another embodiment sequence xix), xxi) or xxiii) corresponds to thesequence from position 1 to position 58 in a sequence selected from thegroup consisting of SEQ ID NO:1253-1632, 1670-1671 and 1673-1678. Inanother embodiment sequence xix), xxi) or xxiii) corresponds to thesequence from position 1 to position 58 in a sequence selected from thegroup consisting of SEQ ID NO:1253-1441, 1670-1671 and 1674-1678 or thegroup consisting of 1442-1632 and 1673.

In one embodiment sequence xix), xxi) or xxiii) corresponds to thesequence from position 1 to position 58 in a sequence selected from thegroup consisting of SEQ ID NO:158, 227, 268, 294, 325, 1176, 1205, 1251,1252, 1270, 1284, 1285, 1298, 1307, 1308, 1311, 1324, 1328, 1330, 1331,1334, 1339, 1340, 1343, 1353, 1361, 1362, 1364, 1367, 1368, 1375, 1393,1415, 1420, 1421, 1422, 1423, 1435, 1471, 1472, 1571, 1594, 1662 and1670-1679, such as from the group consisting of SEQ ID NO:268, 1205,1252, 1270, 1284, 1285, 1298, 1307, 1308, 1324, 1328, 1330, 1331, 1334,1339, 1340, 1343, 1361, 1364, 1367, 1368, 1375, 1393, 1415, 1420, 1421,1422, 1423, 1571, 1670-1672, 1674 and 1676-1679, such as from the groupconsisting of SEQ ID NO:1252, 1285, 1298, 1308, 1324, 1328, 1330, 1331,1340, 1361, 1393, 1415, 1420, 1421, 1670-1672, 1674, 1676-1677 and 1679.

In one particular embodiment sequence xix), xxi) or xxiii) correspondsto the sequence from position 1 to position 58 in a sequence selectedfrom the group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328,1331, 1415, 1421, 1435, 1594 and 1670-1679. In one embodiment sequencexix), xxi) or xxiii) corresponds to the sequence from position 1 toposition 58 in a sequence selected from the group consisting of SEQ IDNO:1252, 1328, 1435 1672, 1675-1676 and 1679, such as the groupconsisting of SEQ ID NO:1252, 1328 and 1679; the group consisting of SEQID NO:1252, 1435 and 1679; or the group consisting of SEQ ID NO:1328 and1435. In one embodiment, sequence xix), xxi) or xxiii) corresponds tothe sequence from position 1 to position 58 in a sequence selected fromthe group consisting of SEQ ID NO:1252, 1328 and 1435.

In one embodiment, sequence xix), xxi) or xxiii) corresponds to thesequence from position 1 to position 58 in a sequence selected from thegroup consisting of SEQ ID NO:1672, 1675-1676 and 1679, such as from thegroup consisting of SEQ ID NO:1672, 1675 and 1676.

In one embodiment, said IL-1R-I binding polypeptide has a meltingtemperature of above 45° C.

Binding of a polypeptide as defined herein to IL-1R-I may interfere withsignaling via IL-1R-I either in vivo or in vitro. Thus, in oneembodiment, there is provided an IL-1R-I binding polypeptide as definedherein which is capable of blocking IL-1R-I dependent signaling.

The half maximal inhibitory concentration (IC₅₀) is a measure of theeffectiveness of a substance for inhibiting a specific quantifiablebiological or biochemical function. This quantitative measure indicateshow much of a particular substance is needed to inhibit a specificbiological function by 50% and is commonly used in the art. In oneparticular embodiment, there is provided an IL-1R-I binding polypeptideas defined herein capable of blocking IL-1R-I signaling such that thehalf maximal inhibitory concentration (IC₅₀) of the blocking is at most1×10⁻⁷ M, such as at most 5×10⁻⁸ M, such as at most 2×10⁻⁸ M, such as atmost 1×10⁻⁸ M, such as at most 5×10⁻⁹ M, such as at most 2×10⁻⁹ M, suchas at most 1×10⁻⁹ M, such as at most 5×10⁻¹⁰ M, such as at most 4×10⁻¹⁰M, such as at most 3×10⁻¹⁰ M. In one embodiment, the half maximalinhibitory concentration (IC₅₀) of the blocking is between 1×10⁻¹⁵ M and1×10⁻⁷ M, such as between 1×10⁻¹⁵ M and 1×10⁻⁸ M, such as between1×10⁻¹⁵ M and 1×10⁻⁹ M, such as between 1×10⁻¹⁵ M and 1×10⁻¹⁰ M, such asbetween 1×10⁻¹⁵ M and 1×10⁻¹¹ M, such as between 1×10⁻¹⁵ M and 1×10⁻¹²M.

The terms “IL-1R-I binding” and “binding affinity for IL-1R-I” as usedin this specification refer to a property of a polypeptide which may betested for example by ELISA, Biolayer Interferometry (BLI) or the use ofsurface plasmon resonance (SPR) technology. For example as described inthe examples below, IL-1R-I binding affinity may be tested in anexperiment in which samples of the polypeptide are captured onantibody-coated ELISA plates and biotinylated IL-1R-I is added followedby streptavidin-conjugated HRP. TMB substrate is added and theabsorbance at 450 nm is measured using a multi-well plate reader, suchas Victor³ (Perkin Elmer). The skilled person may then interpret theresults obtained by such experiments to establish at least a qualitativemeasure of the binding affinity of the polypeptide for IL-1R-I. If aquantitative measure is desired, for example to determine the EC₅₀ value(the half maximal effective concentration) for the interaction, ELISAmay also be used. The response of the polypeptide against a dilutionseries of biotinylated IL-1R-I is measured using ELISA as describedabove. The skilled person may then interpret the results obtained bysuch experiments, and EC₅₀ values may be calculated from the resultsusing for example GraphPad Prism 5 and non-linear regression.

IL-1R-I binding affinity may also be tested in an experiment in whichIL-1R-I, or a fragment thereof, is immobilized on a sensor chip of asurface plasmon resonance (SPR) instrument, and the sample containingthe polypeptide to be tested is passed over the chip. Alternatively, thepolypeptide to be tested is immobilized on a sensor chip of theinstrument, and a sample containing IL-1R-I, or a fragment thereof, ispassed over the chip. The skilled person may then interpret the resultsobtained by such experiments to establish at least a qualitative measureof the binding affinity of the polypeptide for IL-1R-I. If aquantitative measure is desired, for example to determine a K_(D) valuefor the interaction, surface plasmon resonance methods may also be used.Binding values may for example be defined in a Biacore (GE Healthcare)or ProteOn XPR 36 (Bio-Rad) instrument. IL-1R-I is suitably immobilizedon a sensor chip of the instrument, and samples of the polypeptide whoseaffinity is to be determined are prepared by serial dilution andinjected in random order. K_(D) values may then be calculated from theresults using for example the 1:1 Langmuir binding model of theBIAevaluation 4.1 software, or other suitable software, provided by theinstrument manufacturer.

Thus, in one embodiment, there is provided an IL-1R-I bindingpolypeptide as defined herein, which is capable of binding to IL-1R-Isuch that the EC₅₀ value of the interaction is at most 1×10⁻⁷ M, such asat most 5×10⁻⁸ M, such as at most 1×10⁻⁸ M, such as at most 5×10⁻⁹ M,such as at most 1×10⁻⁹ M, such as at most 5×10⁻¹⁰ M, such as at most4×10⁻¹⁰ M, such as at most 3×10⁻¹⁰ M, such as at most 2×10⁻¹⁰ M. In oneembodiment, the EC₅₀ value of the interaction is between 1×10⁻¹⁵ M and1×10⁻⁷ M, such as between 1×10⁻¹⁵ M and 1×10⁻⁸ M, such as between1×10⁻¹⁵ M and 1×10⁻⁹ M, such as between 1×10⁻¹⁵ M and 5×10⁻¹⁰ M, such asbetween 1×10⁻¹⁵ M and 1×10⁻¹⁰ M, such as between 1×10⁻¹⁵ M and 5×10⁻¹¹M, such as between 1×10⁻¹⁵ M and 1×10⁻¹¹ M, such as between 1×10⁻¹⁵ Mand 5×10⁻¹² M, such as between 1×10⁻¹⁵ M and 1×10⁻¹² M.

In one particular embodiment, there is provided an IL-1R-I bindingpolypeptide which is capable of binding to IL-1R-I such that the K_(D)value of the interaction with IL-1R-I is at most 1×10⁻⁶ M, such as atmost 1×10⁻⁷ M, such as at most 5×10⁻⁸ M, such as at most 4×10⁻⁸ M, suchas at most 3×10⁻⁸ M, such as at most 2×10⁻⁸ M, such as at most 1×10⁻⁸ M,such as at most 7×10⁻⁹ M, such as at most 5×10⁻⁹ M, such as at most4×10⁻⁹ M, such as at most 3×10⁻⁹ M, such as at most 2×10⁻⁹ M, such as atmost 1×10⁻⁹ M, such as at most 9×10⁻¹⁰ M, such as at most 7×10⁻¹⁰ M,such as at most 5×10⁻¹⁰ M. In one embodiment, the K_(D) value of theinteraction with IL-1R-I is between 1×10⁻¹⁵ M and 1×10⁻⁶ M, between1×10⁻¹⁵ M and 1×10⁻⁷ M, such as between 1×10⁻¹⁵ M and 1×10⁻⁸ M, such asbetween 1×10⁻¹⁵ M and 1×10⁻⁹ M, such as between 1×10⁻¹⁵ M and 1×10⁻¹⁰ M,such as between 1×10⁻¹⁵ M and 1×10⁻¹¹ M, such as between 1×10⁻¹⁵ M and1×10⁻¹² M.

In one embodiment, there is provided an IL-1R-I binding polypeptide asdefined herein, wherein said IL-1R-I is human IL-1R-I or cynomolgusIL-1R-I, such as human IL-1R-I.

The IL-1R-I binding polypeptide as defined herein may be capable ofblocking the interaction of IL-1R-I with IL-1 cytokines, such as theinteraction of IL-1R-I with IL-1α and/or IL-1β. Thus, in one embodimentsaid IL-1R-I binding polypeptide is capable of blocking the interactionof IL-1R-I with IL-1 cytokines, such as the interaction of IL-1R-I withIL-1α and/or IL-1β.

The skilled person will understand that various modifications and/oradditions can be made to an IL-1R-I binding polypeptide according to anyaspect disclosed herein in order to tailor the polypeptide to a specificapplication without departing from the scope of the present disclosure.

For example, in one embodiment there is provided an IL-1R-I bindingpolypeptide as described herein, which polypeptide has been extended byand/or comprises additional amino acids at the C terminus and/or Nterminus. Such a polypeptide should be understood as a polypeptidehaving one or more additional amino acid residues at the very firstand/or the very last position in the polypeptide chain. Thus, an IL-1R-Ibinding polypeptide may comprise any suitable number of additional aminoacid residues, for example at least one additional amino acid residue.Each additional amino acid residue may individually or collectively beadded in order to, for example, improve production, purification,stabilization in vivo or in vitro, coupling or detection of thepolypeptide. Such additional amino acid residues may comprise one ormore amino acid residues added for the purpose of chemical coupling. Oneexample of this is the addition of a cysteine residue. Additional aminoacid residues may also provide a “tag” for purification or detection ofthe polypeptide, such as a His₆ tag, a (HisGlu)₃ tag (“HEHEHE” tag), a“myc” (c-myc) tag or a “FLAG” tag for interaction with antibodiesspecific to the tag or for immobilized metal affinity chromatography(IMAC) in the case of a His₆-tag.

The further amino acids as discussed above may be coupled to the IL-1R-Ibinding polypeptide by means of chemical conjugation (using knownorganic chemistry methods) or by any other means, such as expression ofthe IL-1R-I binding polypeptide as a fusion protein or joined in anyother fashion, either directly or via a linker, for example an aminoacid linker.

The further amino acids as discussed above may for example comprise oneor more polypeptide domain(s). A further polypeptide domain may providethe IL-1R-I binding polypeptide with another function, such as forexample yet another binding function, an enzymatic function, a toxicfunction, a fluorescent signaling function or combinations thereof.

A further polypeptide domain may moreover provide another IL-1R-Ibinding moiety with the same IL-1R-I binding function. Thus, in afurther embodiment, there is provided an IL-1R-I binding polypeptide ina multimeric form. Said multimer is understood to comprise at least twoIL-1R-I binding polypeptides as disclosed herein as monomer units, theamino acid sequences of which may be the same or different. Multimericforms of the polypeptides may comprise a suitable number of domains,each having an IL-1R-I binding motif, and each forming a monomer withinthe multimer. These domains may have the same amino acid sequence, butalternatively, they may have different amino acid sequences. In otherwords, the IL-1R-I binding polypeptide of the invention may form homo-or heteromultimers, for example homo- or heterodimers. In oneembodiment, there is provided an IL-1R-I binding polypeptide, whereinsaid monomeric units are covalently coupled together. In one embodiment,there is provided an IL-1R-I binding polypeptide, wherein said monomericunits are non-covalently coupled together. In another embodiment, saidIL-1R-I binding polypeptide monomer units are expressed as a fusionprotein. In one embodiment, there is provided an IL-1R-I bindingpolypeptide in dimeric form. One example of an IL-1R-I bindingpolypeptide which will form a disulphide bonded dimer after productionis the polypeptide having the sequence set out in SEQ ID NO:1658. Thehinge region of IgG1 Fc (disclosed in FIG. 1 as position 1-16 of SEQ IDNO:1662) may be used to accomplish dimeric forms of IL-1R-I bindingpolypeptides.

Additionally, “heterogenic” fusion polypeptides or proteins, orconjugates, in which an IL-1R-I binding polypeptide as described herein,or a multimer thereof, constitutes a first domain, or first moiety, andthe second and further moieties have other functions than binding toIL-1R-1, are also contemplated and fall within the ambit of the presentdisclosure. The second and further moiety/moieties of the fusionpolypeptide or conjugate in such a protein suitably also have a desiredbiological activity.

Thus, in a second aspect of the present disclosure, there is provided afusion protein or a conjugate, comprising a first moiety consisting ofan IL-1R-1 binding polypeptide according to the first aspect, and asecond moiety consisting of a polypeptide having a desired biologicalactivity. In another embodiment, said fusion protein or conjugate mayadditionally comprise further moieties, comprising desired biologicalactivities that can be either the same or different from the biologicalactivity of the second moiety.

Thus, in one embodiment, said IL-1R-I binding polypeptide comprises anIL-1R-I binding motif BM as set out in sequence i) or ii); a bindingmodule BMod as set out in sequence iii) or iv), or an amino acidsequence as set out in any one of sequences v)-xxiv). In particular,said IL-1R-I binding motifs may correspond to an amino acid sequencefrom position 8 to 36; said binding modules may correspond to an aminoacid sequence from position 7 to position 55; and said sequence xix),xxi), or xxiii) may be selected from the group consisting of sequencesselected from SEQ ID NO:s shown in FIG. 1 and listed above. Preferredembodiments of IL-1R-I binding polypeptides are disclosed in the firstaspect.

Non-limiting examples of a desired biological activity comprise atherapeutic activity, a binding activity, and an in vivo half-lifeincreasing activity.

In one embodiment, said desired biological activity is an in vivohalf-life increasing activity such that said second moiety increases invivo half-life of the fusion protein or conjugate.

An in vivo half-life increasing activity should in this context beunderstood as an activity which increases the in vivo half-life of thefusion protein or conjugate. The half-life of a first moiety (andpossibly additional moieties) is increased by fusion or conjugation to asecond moiety having an in vivo half-life increasing activity. It willbe appreciated that the corresponding fusion protein or conjugate maycomprise a first IL-1R-I binding moiety as described above, a secondmoiety having an in vivo half-life increasing activity, and optionallyone or more additional moieties, such as an additional IL-1R-I bindingmoiety. For example, said fusion protein or conjugate may comprise twoIL-1R-I binding polypeptides and a second moiety which increases in vivohalf-life compared to the fusion or conjugate comprising the two IL-1R-Ibinding polypeptides only. Examples of fusion proteins comprising anIL-1R-I binding polypeptide and a half-life extending moiety have beenproduced and experimentally tested (Example 10). In one embodiment, saidfusion protein or conjugate comprises a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:1639-1657.

Half-life extension of the resulting fusion protein or conjugate may beaccomplished by a second moiety having an inherently long in vivohalf-life. A polypeptide that increases the hydrodynamic size of thefusion protein or conjugate may for example impose an increasedhalf-life upon the fusion protein or conjugate. Alternatively, thesecond moiety may consist of a natural protein having a long in vivohalf-life, such as serum albumin or the Fc portion of an antibody.

In one embodiment, said second moiety comprises one of the albuminbinding domain of streptococcal protein G or a derivative thereof;albumin; an Fc portion of an antibody, and transferrin. In oneembodiment, said fusion protein or conjugate comprises a sequenceselected from the group consisting of SEQ ID NO:1639-1658.

In one embodiment, said second moiety is an Fc portion of an antibody,such as an IgG1 Fc (SEQ ID NO:1662) or an IgG4 Fc. An IgG4 Fc that maybe used as a half-life extending moiety is the Fc part set out inUniprot, accession number P01861, amino acid residues 99-327. In oneembodiment, said fusion protein or conjugate comprises a polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1646-1654.

In one embodiment, said Fc portion of an antibody is a mutated Fc whichrelative to an unmutated form of Fc displays improved affinity to FcRnand/or reduced effector response. Thus, said Fc portion is a variant ofa naturally occurring Fc in which one or more mutations have beenintroduced. For example, said fusion protein or conjugate comprises apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1734-1737.

In another non-limiting example, wherein said second moiety has in vivohalf-life increasing activity, said second moiety is transferrin. Inparticular embodiments of the fusion protein or conjugate disclosedherein, there is provided a polypeptide selected from the groupconsisting of SEQ ID NO:1657.

In one particular embodiment, the in vivo half-life increasing activityis an albumin binding activity. Non-limiting examples of moietiesproviding such an albumin binding activity are an albumin bindingcamelid Ig V_(HH) domain and an albumin binding engineered single IgV_(H) domain.

In one embodiment, said albumin binding activity is provided by thealbumin binding domain of the peptostreptococcal albumin binding (PAB)protein from Finegoldia magna, or a derivative thereof.

In one embodiment, said albumin binding activity is provided by thealbumin binding domain of streptococcal protein G, or a derivativethereof. Examples of such an albumin binding domain are polypeptideshaving an amino acid sequence as set out in SEQ ID NO:1659-1661.Preferably, the albumin binding domain is as set out in SEQ ID NO:1661.In one embodiment, said fusion protein or conjugate comprises apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1639-1645.

In another non-limiting example, said in vivo half-life increasingactivity is provided by a polyethylene glycol (PEG) or a hydroxyethylstarch (HES).

For example, said fusion protein or conjugate, comprising at least onefurther moiety, may comprise [IL-1R-I binding polypeptide]-[half-lifeincreasing moiety]-[moiety with affinity for selected target]. It is tobe understood that the three moieties in this example may be arranged inany order from the N- to the C-terminal of the polypeptide.

Another non-limiting example is a binding activity which allow thefusion protein to display a modified tissue distribution compared to theunfused IL-1R-I binding polypeptide. In one embodiment the bindingactivity is binding to the transferrin receptor. Such a binding activitycould infer that the conjugate or fusion protein is transferred acrossthe blood brain barrier and into the brain tissue (Yu et al, 2011, SciTransl Med 3(84):84ra44). In a preferred embodiment, such bindingactivity is provided by an antibody fragment.

Another non-limiting example of a binding activity is an activity whichacts to block a biological activity.

In one embodiment, there is provided an IL-1R-I binding polypeptide,fusion protein or conjugate wherein the binding activity acts to block abiological activity.

With regard to the description above of fusion proteins or conjugatesincorporating an IL-1R-I binding polypeptide according to thedisclosure, it is to be noted that the designation of first, second andfurther moieties is made for clarity reasons to distinguish between anIL-1R-I binding polypeptide or polypeptides according to the inventionon the one hand, and moieties exhibiting other functions on the otherhand. These designations are not intended to refer to the actual orderof the different domains in the polypeptide chain of the fusion proteinor conjugate. Similarly, the designations first and second monomer unitsare made for clarity reasons to distinguish between said units. Thus,for example, said first moiety (or monomer unit) may without restrictionappear at the N-terminal end, in the middle, or at the C-terminal end ofthe fusion protein or conjugate.

The skilled person is aware that the construction of a fusion proteinoften involves the use of linkers between the functional moieties to befused, and there are different kinds of linkers with differentproperties, such as flexible amino acid linkers, rigid amino acidlinkers and cleavable amino acid linkers. Linkers have been used to forexample increase stability or improve folding of fusion proteins, toincrease expression, improve biological activity, enable targeting andalter pharmacokinetics of fusion proteins. Thus, in one embodiment, thepolypeptide according to any aspect disclosed herein further comprisesat least one linker, such as at least one linker selected from flexibleamino acid linkers, rigid amino acid linkers and cleavable amino acidlinkers.

In one embodiment, said linker is arranged between a first moietyconsisting of an IL-1R-I binding polypeptide as defined herein and asecond moiety consisting of a polypeptide having a desired biologicalactivity. In another embodiment, said linker is arranged within saidfirst moiety. For example, one or more linker(s) may be arranged betweenmonomeric units of the polypeptide as defined herein. In yet anotherembodiment, linkers may be arranged within said first moiety and betweensaid first moiety and said second moiety.

Flexible linkers are often used in the art when the joined domainsrequire a certain degree of movement or interaction, and may beparticularly useful in some embodiments. Such linkers are generallycomposed of small, non-polar (for example G) or polar (for example S orT) amino acids. Some flexible linkers primarily consist of stretches ofG and S residues, for example (GGGGS)_(p). Adjusting the copy number “p”allows for optimization of linker in order to achieve appropriateseparation between the functional moieties or to maintain necessaryinter-moiety interaction. Apart from G and S linkers, other flexiblelinkers are known in the art, such as G and S linkers containingadditional amino acid residues, such as T and A, to maintainflexibility, as well as polar amino acid residues to improve solubility.In one embodiment, said linker is a flexible linker comprising at leastone amino acid residue(s) selected from the group consisting of glycine,serine and alanine. The skilled person is aware of other suitablelinkers.

In one embodiment, said linker is selected from GS, VDSS, VDGS, VEGS,ASGS, (GGGGS)₂ (SEQ ID NO:1683), AS(GGGGS)₂ (SEQ ID NO:1684) and((KEAAA)₃KELAA)₂ (SEQ ID NO:1685). In one particular embodiment, saidlinker is AS(GGGGS)₂ (SEQ ID NO:1684) or ((KEAAA)₃KELAA)₂ (SEQ IDNO:1685). In one embodiment, said fusion protein or conjugate comprisinga linker comprises a polypeptide having an amino acid sequence as setout in SEQ ID NO:1639-1658. In particular, an exemplary fusion proteinor conjugate comprising the linker AS(GGGGS)₂ is set out in the aminoacid sequence of SEQ ID NO:1646-1658, such as SEQ ID NO:1648 and 1657.

In further aspects of the present disclosure, there is provided apolynucleotide encoding an IL-1R-I binding polypeptide or a fusionprotein as described herein; an expression vector comprising saidpolynucleotide; and a host cell comprising said expression vector.

Also encompassed by this disclosure is a method of producing apolypeptide or fusion protein as described above, comprising culturingsaid host cell under conditions permissive of expression of saidpolypeptide from its expression vector, and isolating the polypeptide.

The IL-1R-I binding polypeptide of the present disclosure mayalternatively be produced by non-biological peptide synthesis usingamino acids and/or amino acid derivatives having protected reactiveside-chains, the non-biological peptide synthesis comprising

-   -   step-wise coupling of the amino acids and/or the amino acid        derivatives to form a polypeptide according to the first aspect        having protected reactive side-chains,    -   removal of the protecting groups from the reactive side-chains        of the polypeptide, and    -   folding of the polypeptide in aqueous solution.

It should be understood that the IL-1R-I binding polypeptide accordingto the present disclosure may be useful as a therapeutic, diagnostic orprognostic agent in its own right or as a means for targeting othertherapeutic or diagnostic agents, with e.g. direct or indirect effectson IL-1R-I. A direct therapeutic effect may for example be accomplishedby inhibiting IL-1R-I signaling.

Thus, in another aspect, there is provided a composition comprising anIL-1R-I binding polypeptide, fusion protein or conjugate as describedherein and at least one pharmaceutically acceptable excipient orcarrier. In one embodiment, said composition further comprises at leastone additional active agent, such as at least two additional activeagents, such as at least three additional active agents. Non-limitingexamples of additional active agents that may prove useful in suchcombination are immune response modifying agents and anti-cancer agentsas described herein. In one embodiment, said composition comprises afusion protein or conjugate wherein said desired biological activity isan in vivo half-life increasing activity such that said second moietyincreases in vivo half-life of the fusion protein or conjugate. Examplesof moieties providing such half-life increasing activities are disclosedelsewhere herein.

Hence, in another aspect of the present disclosure, there is provided anIL-1R-I binding polypeptide, fusion protein, conjugate or composition asdescribed herein for use as a medicament, a prognostic agent or adiagnostic agent. In one embodiment, said IL-1R-I binding polypeptide isprovided for use as a medicament.

In one embodiment, there is provided an IL-1R-I binding polypeptide,fusion protein or conjugate or composition as described herein, for useas a medicament to modulate IL-1R-I function in vivo. As used herein,the term “modulate” refers to changing the activity, such as renderingIL-1R-I function hypomorph, partially inhibiting or fully inhibitingIL-1R-I function.

In one embodiment, there is provided an IL-1R-I binding polypeptide,fusion protein or conjugate or composition as described herein, for use,in the treatment, prognosis or diagnosis of an IL-1R-I related disorder.

As used herein, the term “IL-1R-I related disorder” refers to anydisorder, disease or condition in which IL-1R-I plays a regulatory rolein the signaling pathway. Thus, in one embodiment, there is provided anIL-1R-I binding polypeptide, fusion protein or conjugate or compositionas described herein, for use in the treatment of an IL-1R-I relateddisorder, such as a disorder selected from the group consisting ofinflammatory disease, auto-inflammatory syndrome, autoimmune disease,infectious disease, cardiovascular disease, ischaemic disease, cancerand diabetes. In one embodiment, said IL-1R-I related disorder isselected from the group consisting of inflammatory disease,autoinflammatory syndrome and autoimmune disease.

In one embodiment, said IL-1R-I related disorder is selected from thegroup consisting of familial Mediterranean fever (FMF);cryopyrin-associated periodic syndrome (CAPS); TNF receptor-associatedperiodic syndrome (TRAPS); hyper-IgD syndrome (HIDS); periodic fever;aphthous stomatitis; pharyngitis; adenitis (PFAPA); rheumatoid arthritis(RA), juvenile RA, juvenile idiopathic arthritis, systemic juvenileidiopathic arthritis; adult-onset Still's disease; Schnitzler syndrome;Muckle Wells Syndrome; macrophage activation syndrome; Behget's disease;uveitis; acne vulgaris; pyoderma gangrenosum; gout; type 2 diabetes,new-onset diabetes; dry eye syndrome; hidradenitis suppurativa;neutrophilic dermatoses, in particular cytophagic histiocyticpanniculitis, Weber-Christian disease, and neutrophilic panniculitis;cardiovascular disease, myocardial infarction, stroke; liver failure,kidney failure; acute lung injury; pseudogout, calcium-pyrophosphatedeposition disorder, chondrocalcinosis, deficiency of the IL-1 receptorantagonist (DIRA), deficiency of the IL-36 receptor antagonist (DITRA),ADAM2 deficiency (DADA2), pyogenic arthritis, pyoderma gangrenosum andacne (PAPA) syndrome, pyoderma gangrenosum, acne and suppurativehidradenitis (PASH) syndrome, PAPA and suppurative hidradenitis (PAPASH)syndrome, autoinflammatory syndrome with lymphedema (AISLE),Phospholipase C-gamma-2 mutation autoinflammatory syndrome,NALP12-associated periodic syndrome (NAPS12), mevalonate kinasedeficiency (MKD), psoriatic arthritis, reactive arthritis, ankylosingspondylitis, haemochromatosis-related arthritis, periarticularcalcinosis, osteoarthritis, inflammatory osteoarthritis, handosteoarthritis, pustular psoriasis such as generalised pustularpsoriasis (GPP), palmoplantar pustulosis (PPP), acrodermatitis continuaof Hallopeau (ACH); Blau syndrome; Sweet syndrome; various vasculitidessuch as giant-cell arteritis (GCA), polymyalgia rheumatica (PMR),Takayasu arteritis, Kawasaki disease, urticarial vasculitis, andHenoch-Schönlein purpura (HSP); neutrophilic urticaria and idiopathiccold urticaria; lichen planus; polymyositis, dermatomyositis, juveniledermatomyositis and inclusion-body myositis; various stages of myelomasuch as smoldering myeloma (SMM), indolent myeloma, Waldenstrom'smacroglobulinemia, and multiple myeloma; tumor-induced cachexia; solidtumor growth; neonatal disorders such as bronchopulmonary dysplasia(prophylaxis), necrotising enterocolitis (NEC), retinopathy ofprematurity (ROP), cerebral palsy due to perinatal cerebral ischemia,and infant respiratory distress syndrome (IRDS); Whipple's disease;traumatic brain injury; refractory epilepsy; systemic inflammatoryresponse syndrome (SIRS); cutaneous lupus; Jessner-Kanof disease;amyotrophic lateral sclerosis; systemic sclerosis (scleroderma); septicshock; acute pancreatitis; chronic recurrent multifocal osteomyelitis,non-bacterial osteitis (NBO), synovitis, acne, pustulosis, hyperostosis,osteitis (SAPHO) syndrome, and Majeed syndrome; relapsingpolychondritis; idiopathic recurrent pericarditis (IRP); myocarditis;Erdheim-Chester disease; juvenile xantogranuloma; islet-celltransplantation; haemodialysis-induced systemic inflammation;graft-versus-host disease; ANCA-associated glomerulonephritis andrecurrent glomerulonephritis; Cogan syndrome; autoimmune inner-eardisease; chronic granulomatous disease (CGD); Castleman's disease;cardiac failure; diastolic cardiac failure; antisynthetase syndrome;acute ACL injury; acute haemorrhagic leukoencephalitis; AA amyloidosis;Di George syndrome; generalised fatigue; chronic fatigue syndrome (CFS);gulf-war illness (GWI); and narcolepsy.

In another embodiment, said IL-1R-I related disorder is cancer, such asa cancer selected from the group consisting of colon cancer, breastcancer, lung cancer, head and neck cancer, melanoma and prostate cancer.

The skilled person will appreciate that said IL-1R-I bindingpolypeptide, fusion protein or conjugate or a composition comprising ananti-IL-1R-I binding polypeptide, fusion protein or conjugate asdescribed herein may be administered to a subject using standardadministration techniques, including oral, topical, intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, duodenal or suppository administration.Thus, in one embodiment, there is provided an IL-1R-I bindingpolypeptide, fusion protein or conjugate or a composition as describedherein for oral, topical, intravenous, intraperitoneal, subcutaneous,pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual,duodenal or suppository administration. In one embodiment, saidadministration is oral, intravenous, subcutaneous or duodenaladministration and in one particular embodiment, said administration isoral administration.

As used herein, the term “subject” refers to a mammalian subject, suchas a human subject.

Non-limiting examples of indications where topical administration routescould be especially relevant in IL-1R-I mediated disease include oculardiseases such as dry eye disease, uveitis, scleritis, and ulcerativekeratitis; dermatological diseases such as ichtyosis and generalizedpustular psoriasis; pulmonary diseases such as adult respiratorydistress syndrome, bronchopulmonary dysplasia, chronic obstructivepulmonary disease, asbestosis, and silicosis.

In some embodiments it may be beneficial that the polypeptide, fusionprotein, conjugate or composition for use as described above, isadministered repeatedly within a period of time, for example within a 24hour period from disease onset. In one particular embodiment whereinsaid IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition is administered repeatedly within a 24 hour period fromdisease onset, such as at least two times within a 24 hour period fromdisease onset, such as at least three times within a 24 hour period fromdisease onset, to a subject in need thereof. In yet a particularembodiment, said IL-1R-I binding polypeptide, fusion protein, conjugateor composition is administered continuously during a limited time periodsuch as during 24 hours from disease onset. Repeated or continuousadministration may be preferred when distribution to the brain isrequired, such as in acute treatment of stroke.

In some embodiments wherein said fusion protein or conjugate comprises asecond moiety having an in vivo half-life increasing activity, saidfusion protein or conjugate, or composition comprising such a fusionprotein or conjugate, is administered once weekly to a subject in needthereof.

In a related aspect, there is provided a method of treatment of anIL-1R-I related disorder, comprising administering to a subject in needthereof an effective amount of an IL-1R-I binding polypeptide, fusionprotein, conjugate or a composition as described herein. In a morespecific embodiment of said method, the IL-1R-I binding polypeptide,fusion protein, conjugate or composition as described herein modulatesIL-1R-I function in vivo.

In one embodiment, said IL-1R-I related disorder is selected from thegroup as defined above in connection with the previous aspect.

In some embodiments of said method of treatment, it may be beneficialthat the polypeptide, fusion protein, conjugate or composition asdescribed above, is administered repeatedly within a period of time, forexample within a 24 hour period from disease onset. In one particularembodiment wherein said IL-1R-I binding polypeptide, fusion protein,conjugate or composition is administered repeatedly within a 24 hourperiod from disease onset, such as at least two times within a 24 hourperiod from disease onset, such as at least three times within a 24 hourperiod from disease onset, to a subject in need thereof. In yet aparticular embodiment, said IL-1R-I binding polypeptide, fusion protein,conjugate or composition is administered continuously during a limitedtime period, such as within 24 hours from disease onset. Repeated orcontinuous administration may be preferred when distribution to thecentral nervous system is required, such as in acute treatment ofstroke.

In other embodiments, of said method of treatment, said fusion proteinor conjugate comprises a second moiety having an in vivo half-lifeincreasing activity, or said composition comprises a fusion protein orconjugate comprising a second moiety having an in vivo half-lifeincreasing activity. In such methods, said fusion protein, conjugate orcomposition may be administered once weekly to a subject in needthereof.

It may be beneficial to administer a therapeutically effective amount ofan IL-1R-I binding polypeptide, fusion protein or conjugate orcomposition as described herein and at least one second drug substance,such as an immune response modulating agent as described above or ananti-cancer agent.

As used herein, the term “co-administration” encompasses bothconcomitant and sequential administration. Thus, in one embodiment thereis provided a method as defined above further comprisingco-administration of an immune response modulating agent as describedabove. In another embodiment there is provided a method as defined abovefurther comprising co-administration of an anti-cancer agent asdescribed above.

In another aspect of the present disclosure, there is provided a methodof detecting IL-1R-I, comprising providing a sample suspected to containIL-1R-I, contacting said sample with an IL-1R-I binding polypeptide,fusion protein, conjugate or a composition as described herein, anddetecting the binding of the IL-1R-I binding polypeptide, fusionprotein, conjugate or composition to indicate the presence of IL-1R-I inthe sample. In one embodiment, said method further comprises anintermediate washing step for removing non-bound polypeptide, fusionprotein, conjugate or composition, after contacting the sample.

While the invention has been described with reference to variousexemplary aspects and embodiments, it will be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted for elements thereof without departing from the scope ofthe invention. In addition, many modifications may be made to adapt aparticular situation or molecule to the teachings of the inventionwithout departing from the essential scope thereof. Therefore, it isintended that the invention not be limited to any particular embodimentcontemplated, but that the invention will include all embodimentsfalling within the scope of the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a listing of the amino acid sequences of examples of IL-1R-Ibinding polypeptides of the present disclosure (SEQ ID NO:1-1638 and1667-1679), albumin binding polypeptides (SEQ ID NO:1659-1661); humanIgG1 Fc (SEQ ID NO:1662), human albumin (SEQ ID NO:1663) and humantransferrin (SEQ ID NO:1664), examples of IL-1R-1 binding fusionpolypeptides comprising said polypeptides (SEQ ID NO:1639-1658 and1734-1737) optionally including a linker, control polypeptides(GS-hIL-1β, SEQ ID NO:1733) as well as the amino acid sequences ofcynomolgus IL-1R-I-His₆ (SEQ ID NO:1665) and cynomolgus IL-1R-1-Fc (SEQID NO:1666) used for selection, screening and/or characterization forillustration of the invention. The deduced IL-1R-I binding motifs (BMs)of the IL-1R-I binding polypeptides disclosed herein extend from residue8 to residue 36 in sequences with SEQ ID NO:1-1638 and 1667-1668 and1670-1679. In SEQ ID NO:1669, said BM extends from residue 5 to residue33. The amino acid sequences of the 49 amino acid residues longpolypeptides (BMod) predicted to constitute the complete three-helixbundle within each of these Z variants extend from residue 7 to residue55, except for the N-terminally truncated Z variant with SEQ ID NO:1669wherein it extends from residue 4 to residue 52.

FIG. 2 is a diagram showing concentration-response curves for inhibitionof IL-1β induced IL-6 release in NHDF cells for six anti-IL-1R-I bindingfusion proteins (PS10536, SEQ ID NO:1648; PS10537, SEQ ID NO:1649;PS10534, SEQ ID NO:1646; PS10535, SEQ ID NO:1647; PS10538, SEQ IDNO:1650; PS10539, SEQ ID NO:1651). Anakinra (dotted line) was includedas a reference.

FIG. 3 is a diagram showing concentration of IL-1R-I binding Z variantsat 50% inhibition (IC₅₀) of IL-1β induced IL-6 release in human wholeblood from five to ten individual donors. The median IC₅₀ was 0.31, 1.25and 0.30 nM for PS10536 (SEQ ID NO:1648), PS1537 (SEQ ID NO:1649) andanakinra, respectively.

EXAMPLES Summary

The following Examples disclose the development of novel Z variantmolecules targeted to interleukin 1 receptor I (IL-1R-1) based on phagedisplay technology. The IL-1R-I binding polypeptides described hereinwere sequenced, and their amino acid sequences are listed in FIG. 1 withthe sequence identifiers SEQ ID NO:1-1632. Additional IL-1R-I bindingpolypeptides disclosed herein are listed in FIG. 1 with sequenceidentifiers SEQ ID NO:1633-1638 and 1667-1679. The Examples furtherdescribe the characterization of IL-1R-I binding polypeptides and invitro functionality of said polypeptides. As used herein, the term“IL-1R-I binding Z variants” refers to 58 amino acid long IL-1R-Ibinding polypeptides comprising an IL-1R-I binding motif as disclosedherein.

Example 1 Selection and Screening of IL-1R-I Binding Z Variants

In this Example, human and cynomolgus IL-1R-I (hIL-1R-I and cIL-1R-1,respectively) were used as target proteins in phage display selectionsusing a phage library of Z variants. The DNA of selected clones wassequenced, the Z variants were produced in E. coli as periplasmicfractions and assayed against IL-1R-I in ELISA (enzyme-linkedimmunosorbent assay).

Materials and methods

Biotinylation of target protein: Human IL-1R-1-Fc (hIL-1 RI-Fc CreativeBioMart cat. no. IL1 RI-771H) and human IL-1R-I (hIL-1 RI, RnD Systemscat. no. 269-1R/CF) were biotinylated according to the manufacturer'srecommendations at room temperature (RT) for 30 min using No-WeighEZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific, cat. no. 21327) at a10×molar excess. Prior to the biotinylation, a buffer exchange tophosphate buffered saline (PBS, 10 mM phosphate, 137 mM NaCl, 2.68 mMKCl, pH 7.4) was performed for hIL-1R-1-Fc using a dialysis cassette(Slide-a-lyzer 3.5 K, 3500 MWCO, Thermo Scientific, cat. no. 66333),according to the manufacturer's instructions, and hIL-1R-I was dissolvedin 100 mM phosphate buffer pH 6.5. After the biotinylation, a bufferexchange to PBS was performed for both proteins, as described above.

Expression of his-tagged target protein: Expression of cynomolgusIL-1R-I-His₆(cIL-1R-I-His₆, SEQ ID NO:1665) was performed using theFreeStyle 293-F expression system (Thermo Fisher Scientific),essentially according to the manufacturer's protocol. Supernatants wereharvested by centrifugation 5 days after transfection of expressionvectors and stored at −70° C. The frozen supernatant from the FreeStyle293-F cultures were thawed and filtrated (0.22 μm). The supernatant,containing the cIL-1R-I-His₆ was purified using affinity chromatographywith an IMAC column. The purified protein was buffer exchanged to PBS.The purity of the protein was analyzed by SDS-PAGE stained withCoomassie Blue and the molecular weight was analyzed using massspectrometry (HPLC/MS or MALDI-TOF/MS).

Phage display selection of IL-1R-I binding Z variants: A library ofrandom variants of protein Z displayed on bacteriophage in fusion to awild type albumin binding domain (abbreviated ABD001, SEQ ID NO:1660)was used to select IL-1R-I binding Z variants. The library, which isdenoted Zlib006Naive.II, has a size of 1.5×10¹⁰ library members (Zvariants). The construction in the phage library vector pAY02592 and thephage stock production was previously described in WO 2016/113246.

Selections against hIL-1R-I-Fc, biotinylated hIL-1R-1-Fc(b-hIL-1R-1-Fc), biotinylated hIL-1R-I (b-hIL-1R-1) and cIL-1R-I-His₆(SEQ ID NO:1665) were performed in four selection cycles. In the firstcycle, four selection tracks were run in parallel; 1-1 to 1-4. Theselection tracks 1-1 and 1-4 were divided into two parts in cycle two,resulting in a total of six parallel tracks in round 2, 3 and 4; 2-1 to2-9, 3-1 to 3-9 and 4-1 to 4-9, respectively, as presented in Table 2.The number of phage particles used for selections was more than 2000times the number of eluted phage particles in the previous cycle, but alower amount was used in selection tracks 4-1 to 4-5. Phage stockpreparation, selection procedure and amplification of phage betweenselection cycles were performed essentially as described in WO2009/077175 with the following exceptions 1-8. Exception 1: PBSsupplemented with 10% fetal calf serum (FCS, Gibco, cat. no. 10108-165),1.5 μM human serum albumin (HSA, Novozymes, cat. no. 230-005) and 0.1%Tween20 (Acros Organics, cat. no. 233362500) was used as selectionbuffer. Exception 2: pre-selection was performed in cycles 1-4 at >60min at RT or overnight at 4° C. by incubation of phage stock withDynabeads® M-280 Streptavidin (SA beads, Life technologies/Invitrogen,cat. no. 11206D), SA beads pre-coated with biotinylated human IgG1-Fc(b-hFc, Jackson Immuno Research cat. no. 009-060-008) (Fc/SA beads), SAbeads pre-coated with b-hFc and (Z00000)₂-Cys-biotin, preparedessentially as described in WO 2009/077175 (Fc/Z00000/SA beads), proteinA beads (Life technologies/Novex, cat. no. 10002D) pre-coated with b-hFc(Fc/protein A beads) or Dynabeads® His-Tag Isolation and Pulldown (IMbeads, Life Technologies/Novex, cat. no. 10104D), respectively, aspresented in Table 2. b-hFc was incubated with the beads (5, 8.5 or 10μg b-hFc per mg Z00000/SA beads, protein A beads or SA beads,respectively) for >1 h at RT, then the beads were washed with 2×PBST(PBS supplemented with 0.1% Tween20) before use in pre-selection.Exception 3: all tubes and beads used in the selections were pre-blockedwith PBS supplemented with 3% BSA (bovine serum albumin, Sigma cat. no.A3059) and 0.1% Tween20. Exception 4: selections were performed insolution at RT and the time for selection was 140 min in the first cycleand 120 min in the following cycles. Exception 5: target-phage complexeswere captured on beads using the following amount of target protein andbeads: 3.2 μg hIL-1R-1-Fc per mg Z00000/SA beads, 4 μg hIL-1R-1-Fc permg protein A beads, 2 μg b-hIL-1R-1-Fc or 2 μg b-hIL-1R-I per mg SAbeads and 4.5 μg cIL-1R-I-His₆ per mg IM beads, respectively. The beadtype used for each selection track is the same as presented forpre-selection in Table 2. Exception 6: E. coli strain ER2738 cells(Lucigen, Middleton, Wis., USA) grown in medium supplemented with 10μg/ml tetracycline were used for phage infection. Exception 7: a 5×excess of M13K07 helper phage compared to bacteria was allowed to infectlog phase bacteria. Exception 8: amplification of phage particles afterround 2 and 3 was performed essentially as described in WO 2016/113246;Example 5, but using Tryptic Soy Broth+Yeast Extract (TSB-YE) mediumsupplemented with 100 μM IPTG, 25 μg/ml kanamycin, 100 μg/ml ampicillinand 2% glucose in the overnight culture.

An overview of the selection strategy, describing an increasedstringency in the selection cycles obtained by using a lowered targetconcentration and an increased number of washes, is shown in Table 2.

TABLE 2 Overview of the phage display selection from a naive library.Phage stock Target Number Selection from library or Preselectionconcentration of Cycle track selection track beads Target (nM) washes 11-1 Zlib006Naive.II Fc/Z00000/SA hIL-1R-I-Fc 100 2 1 1-2 Zlib006Naive.IIFc/protein A hIL-1R-I-Fc 100 2 1 1-3 Zlib006Naive.II Fc/SA b-hIL-1R-I-Fc100 2 1 1-4 Zlib006Naive.II SA b-hIL-1R-I 100 2 2 2-1 1-1 Fc/Z00000/SAhIL-1R-I-Fc 67 4 2 2-2 1-1 Fc/Z00000/SA hIL-1R-I-Fc 33 4 2 2-3 1-2Fc/protein A hIL-1R-I-Fc 67 4 2 2-5 1-3 Fc/SA b-hIL-1R-I-Fc 67 4 2 2-71-4 SA b-hIL-1R-I- 67 4 2 2-9 1-4 IM cIL-1R-I-His₆ 67 4 3 3-1 2-1Fc/Z00000/SA hIL-1R-I-Fc 44 6 3 3-2 2-2 Fc/Z00000/SA hIL-1R-I-Fc 11 6 33-3 2-3 Fc/protein A hIL-1R-I-Fc 44 6 3 3-5 2-5 Fc/SA b-hIL-1R-I-Fc 44 63 3-7 2-7 SA b-hIL-1R-I 44 6 3 3-9 2-9 SA b-hIL-1R-I 44 6 4 4-1 3-1Fc/Z00000/SA hIL-1R-I-Fc 30 8 4 4-2 3-2 Fc/Z00000/SA hIL-1R-I-Fc 3.7 8 44-3 3-3 Fc/protein A hIL-1R-I-Fc 30 8 4 4-5 3-5 Fc/SA b-hIL-1R-I-Fc 30 84 4-7 3-7 SA b-hIL-1R-I 30 8 4 4-9 3-9 IM cIL-1R-I-His₆ 30 8

Washes were performed for 1 min using PBST 0.1%, and elution was carriedout as in the protocol for bead bound phage/target complexes eluted atpH 2 described in WO 2009/077175.

Production of Z variants for ELISA: The Z variants were producedessentially as described in WO 2016/113246 with the exception that thefinal volume of pellet dissolving from the 1 ml culture was 1000 μl PBST0.05% (PBS supplemented with 0.05% Tween20).

The final supernatant of the periplasmic extract contained the Zvariants as fusions to ABD (Z-ABD; ABD001, SEQ ID NO:1660), expressed asAQHDEALE-[Z#####]-VDYV-[ABD]-YVPG (Gronwall et al. (2007) J Biotechnol,128:162-183). Z##### refers to individual 58 amino acid residue Zvariants.

ELISA screening of Z variants: The binding of Z variants to humanIL-1R-I was analyzed in ELISA, essentially as described in WO2016/113246, using 1 nM hIL-1R-1-Fc as target, and using a goatanti-human IgG-HRP (Southern Biotech, cat. no. 2040-05) diluted 1:10,000for detection. As blank control, PBST 0.05% was added instead of theZ-ABD periplasmic sample and as negative control, a periplasmic extractwith ABD001 (SEQ ID NO:1660) was added instead of the Z-ABD periplasmicsample. Control plates were assayed in a similar setup using 100 nMb-hFc instead of target protein and streptavidin conjugated HRP (ThermoScientific, cat. no. N100) diluted 1:30,000 for detection.

Sequencing: In parallel with the ELISA screening, the clones were pickedfor sequencing. PCR fragments, amplified from single colonies, weresequenced and analyzed essentially as described in WO 2009/077175.

EC₅₀ analysis of Z variants: A selection of IL-1R-I binding Z variantswas subjected to an analysis of response against a dilution series ofhIL-1R-1-Fc using ELISA as described above. The target proteinhIL-1R-1-Fc was diluted stepwise 1:10 from 100 to 0.01 nM. As abackground control, the Z variants were assayed with no target proteinadded. Obtained data was analyzed using GraphPad Prism 5 and non-linearregression, and the EC₅₀ values (the half maximal effectiveconcentration) were calculated.

Results

Phage display selection of IL-1R-I binding Z variants: Individual cloneswere obtained after four cycles of phage display selections againsthuman and cynomolgus IL-1R-1.

ELISA screening of Z variants: The clones obtained after four cycles ofselection were produced individually in 96-well plates and were screenedfor hIL-1R-1-Fc binding activity in ELISA using 1 nM target protein. Inparallel, an ELISA to exclude Fc binding was run. Clones positive tohIL-1R-I were identified and none of the IL-1R-I positive clones werepositive to Fc solely. No response was obtained for the ABD negativecontrol. 45 identified Z variants displayed an ELISA screening resultthat was more than 0.71 AU (8.5× the control background).

Sequencing: In parallel with ELISA screening, sequencing was performedfor clones obtained after four cycles of selection. Each variant wasgiven a unique identification number #####, and individual variants arereferred to as Z#####. The amino acid sequences of a subset of theidentified 58 amino acid residues long Z variants are listed in FIG. 1and in the sequence listing as SEQ ID NO:1-19. The deduced IL-1R-Ibinding motifs extend from residue 8 to residue 36 in each sequence. Theamino acid sequences of the 49 amino acid residues long polypeptidespredicted to constitute the complete three-helix bundle within each ofthese Z variants extend from residue 7 to residue 55.

EC₅₀ analysis of Z variants: A subset of said Z variants (SEQ IDNO:1-19) was subjected to an ELISA target titration using hIL-1R-I-Fc.Obtained results were used for calculation of EC₅₀ values (Table 3).

TABLE 3 Calculated EC₅₀ values from ELISA titration analysis. Z variantSEQ ID NO EC₅₀ (M) Z12884 13 6.4 × 10⁻¹⁰ Z12891 19 2.2 × 10⁻⁹  Z12895 122.3 × 10⁻¹⁰ Z12905 7 7.3 × 10⁻⁹  Z12910 11 2.3 × 10⁻¹⁰ Z12911 18 3.2 ×10⁻¹⁰ Z12912 10 3.9 × 10⁻¹⁰ Z12915 5 4.3 × 10⁻⁹  Z12917 15 3.3 × 10⁻¹⁰Z12920 14 6.6 × 10⁻⁹  Z12922 3 2.9 × 10⁻⁹  Z12929 17 1.7 × 10⁻⁸  Z129332 1.5 × 10⁻⁹  Z12941 4 4.6 × 10⁻¹⁰ Z12961 16 2.9 × 10⁻¹⁰ Z12964 1 3.9 ×10⁻¹⁰ Z12967 9 1.7 × 10⁻¹⁰ Z12974 8 2.1 × 10⁻¹⁰ Z12975 6 2.6 × 10⁻¹⁰

Example 2 Production and Characterization of Primary IL-1R-1 Binding ZVariants

This Example describes the general procedure for subcloning andproduction of His-tagged Z variants originating from the primary phageselection, characterization of their target binding, target blocking andmelting points.

Materials and Methods

Subcloning of Z variants with a His₆ tag: The DNA of respective Zvariant was amplified from the phage library vector pAY02592. Asubcloning strategy for construction of monomeric Z variant moleculeswith N-terminal His₆ tag was applied using standard molecular biologytechniques. The Z gene fragments were subcloned into an expressionvector resulting in the encoded sequence MGSSHHHHHHLQ-[Z#####]-VD.

Cultivation: E. coli T7E2 cells (GeneBridges) were transformed withplasmids containing the gene fragment of each respective IL-1R-I bindingZ variant. The resulting recombinant strains were cultivated in mediasupplemented with 50 μg/ml kanamycin, either at 30° C. in 50 ml scaleusing the EnPresso protocol (BioSilta), or at 37° C. in 930 ml TSB-YEmedium. In order to induce protein expression, IPTG was added to a finalconcentration of 0.2 mM at OD600≈10 (EnPresso) or 2 (TSB+YE). Afterinduction, the cultivations were incubated for 16 h (EnPresso) or 5 h(TSB+YE). The cells were harvested by centrifugation.

Purification of IL-1R-I binding Z variants with a His₆ tag:Approximately 1-2 g of each cell pellet was resuspended in bindingbuffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4)supplemented with Benzonase® (Merck). After cell disruption, cell debriswas removed by centrifugation and each supernatant was applied on a 1 mlHis GraviTrap IMAC column (GE Healthcare). Contaminants were removed bywashing with wash buffer (20 mM sodium phosphate, 0.5 M NaCl, 60 mMimidazole, pH 7.4) and the Z variants were subsequently eluted withelution buffer (20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH7.4). After the IMAC purification, the buffer was exchanged to PBS (2.68mM KCl, 137 mM NaCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH 7.4) using PD-10desalting columns (GE Healthcare). All Z variants were subjected to asecond purification step. Each Z variant was loaded on a 1 ml Resource15RPC column (GE Healthcare), previously equilibrated with RPC solvent A(0.1% TFA, 10% ACN, 90% water). After column wash with RPC solvent A,bound proteins were eluted with a linear gradient 0-50% RPC solvent B(0.1% TFA, 80% ACN, 20% water) for 20 ml. The buffer was then exchangedto PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH₂PO₄, 8.1 mM Na₂HPO₄, pH7.4) using PD-10 desalting columns (GE Healthcare).

Protein concentrations were determined by measuring the absorbance at280 nm, using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB)and the extinction coefficient of the respective protein. Samples with aconcentration less than approximately 1 mg/ml were concentrated usingAmicon Ultra-4, Ultracel-3K (MerckMillipore). The purity was analyzed bySDS-PAGE stained with Coomassie Blue and the identity of each purified Zvariant was confirmed using LC/MS analysis.

Biacore affinity analysis: Affinities (K_(D)) for human and cynomolgusIL-1R-I were determined for His₆-tagged Z variants using a Biacore 2000instrument (GE Healthcare). hIL-1R-I or cIL-1R-I-His₆ was immobilized onthe carboxylated dextran layer in different flow cells of a CM5 chip (GEHealthcare, cat. no. BR100012). The immobilization was performed usingamine coupling chemistry according to the manufacturer's protocol.Acetate pH 4.5 (GE Healthcare, cat. no. BR100350) was used for liganddilution and HBS-EP (GE Healthcare, cat. no. BR100188) was used asrunning buffer. One flow cell surface on the chip was activated anddeactivated for use as blank during analyte injections. In the kineticexperiment, HBS-EP was used as running buffer, the flow rate was 50μl/min and the temperature was 25° C. The analytes, i.e. the Z variants,were each diluted in HBS-EP buffer to final concentrations of 40 nM and10 nM, and were injected over the target chip surfaces for 2 min,followed by dissociation in running buffer for 8 min. Kinetic constantswere calculated from the obtained sensorgrams using a 1:1 binding model,in the BiaEvaluation software 4.1 (GE Healthcare).

In vitro IL-1β neutralization assay: The TF-1 cell line proliferates inresponse to many different cytokines including IL-1β. This cell line wasused to assess the ability of primary IL-1R1 binders to inhibit IL-1βactions. TF-1 cells were maintained in RPM11640 with L-glutamine (Lonza)supplemented with 10% FCS (Gibco), Penicillin-Streptomycin (Lonza) and 2ng/ml rhGM-CSF (R&D Systems). Prior to use, cells were washed twice inRPM11640 without rhGM-CSF. Cells were then counted and dispensed into 96well flat bottomed plates at a density of 3×10⁴ cells per well. Inseparate plates, serial dilutions of the inhibitory binders; His₆-taggedIL-1R-I binding Z variants, with a concentration range of 400-0.1 nM, orthe IL-1R-I binding protein Kineret (anakinra, Sobi), with aconcentration range of 0.3-0.0003 nM, were incubated in the presence of0.6 nM IL-1β (PeproTech). The pre-mixed complexes of the Z variantpolypeptides and IL-13 were transferred to wells containing TF-1 cells.The cells were stimulated for 72 h at 37° C. in a humidified 5% CO₂atmosphere. During the last four hours of incubation, 19 μl of 2×diluted CCK-8 (Fluka, Sigma Aldrich), was added per well to determinethe proliferative responses. The absorbance was measured at 450 nm usinga microplate reader (Victor3, Perkin Elmer). The data on cell growth wasassessed by non-linear regression to a four-parameter dose-responsecurve, and the half maximal inhibitory concentration (IC₅₀) wasdetermined using GraphPadPrism program.

Circular dichroism (CD) spectroscopy analysis: Purified His₆-tagged Zvariants were diluted to 0.5 mg/ml in PBS. For each diluted Z variant, aCD spectrum at 250-195 nm was obtained at 20° C. In addition, a variabletemperature measurement (VTM) was performed to determine the meltingtemperature (Tm). In the VTM, the absorbance was measured at 221 nmwhile the temperature was raised from 20 to 90° C., with a temperatureslope of 5° C./min. A new CD spectrum was obtained at 20° C. after theheating procedure in order to study the refolding ability of the Zvariants. The CD measurements were performed on a Jasco J-810spectropolarimeter (Jasco Scandinavia AB) using a cell with an opticalpath length of 1 mm.

Results

Production of His₆-tagged Z variants: The IL-1R-I binding Z variantswith a His₆ tag were expressed as soluble gene products in E. coli. Theamount of purified protein from approximately 1-2 g bacterial pellet wasdetermined spectrophotometrically by measuring the absorbance at 280 nmand ranged from approximately 3 to 25 mg for the different IL-1R-Ibinding Z variants before RPC purification. SDS-PAGE analysis of eachfinal protein preparation showed that these predominantly contained theIL-1R-I binding Z variant. The correct identity and molecular weight ofeach Z variant were confirmed by HPLC-MS analysis.

Biacore affinity analysis: The interactions of His₆-taggedIL-1R-1-binding Z variants with human and cynomolgus IL-1R-I wereanalyzed in a Biacore instrument by injecting two concentrations of eachZ variant over surfaces containing immobilized IL-1R-1. The calculatedaffinities (K_(D)) for human IL-1R-I are presented in Table 4. Thestrongest binder to human IL-1R-I (Z12967, SEQ ID NO:9) bound to humanIL-1R-I with an affinity of 1.2×10⁻⁹ M and to cynomolgus IL-1R-I with anaffinity of 3.5×10⁻⁸ M.

TABLE 4 Affinities for His₆-Z polypeptides binding to IL-1R-I. Z variantSEQ ID NO Human IL-1R-I K_(D) (M) Z12884 13 1.8 × 10⁻⁸ Z12895 12 5.5 ×10⁻⁹ Z12911 18 7.1 × 10⁻⁹ Z12917 15 8.0 × 10⁻⁹ Z12922 3 9.8 × 10⁻⁹Z12967 9 1.2 × 10⁻⁹ Z12974 8 1.5 × 10⁻⁸ Z12975 6 8.9 × 10⁻⁸

In vitro IL-1β neutralization assay: The IL-1β inhibition ability ofHis₆-tagged IL-1R-I-binding Z variants was analyzed in a TF-1 cellassay. The resulting IC₅₀ values are presented in Table 5.

TABLE 5 IC₅₀ values for His₆-Z polypeptides from a TF-1 cell assay. Zvariant SEQ ID NO IC₅₀ (nM) Z12884 13 27 Z12895 12 7.1 Z12911 18 30Z12917 15 11 Z12922 3 66 Z12967 9 1.1 Z12974 8 60 Z12975 6 35 Kineret —0.05

CD analysis: The CD spectra determined for the IL-1R-I binding Zvariants with a His₆ tag showed that each had an a-helical structure at20° C. This result was also verified in the variable temperaturemeasurements, wherein the melting temperatures were determined (Table6). Reversible folding was seen for all the IL-1R-I binding Z variantswhen overlaying spectra measured before and after heating to 90° C.

TABLE 6 Melting temperatures (Tm). Z variant SEQ ID NO Tm (° C.) Z1291118 64 Z12967 9 58 Z12922 3 47 Z12974 8 49 Z12975 6 51 Z12884 13 49Z12917 15 56 Z12895 12 55

Example 3 Design and Construction of a First Matured Library of IL-1R-1Binding Z Variants

In this Example, a matured library was constructed. The library was usedfor selection of further IL-1R-I binding Z variants. Selections frommatured libraries are usually expected to result in binders withincreased affinity (Orlova et al., (2006) Cancer Res 66(8):4339-48).

Materials and Methods

Library design: The library was primarily based on human IL-1R-I bindingZ variants from the primary selection described in Example 1 and 2.Sequences of a subset of the Z variants derived from the primaryselection are listed in FIG. 1 and in the sequence listing as SEQ IDNO:1-19. In the new library, denoted Zlib0061L-1 RI.I, ten variablepositions in the Z molecule scaffold were biased towards certain aminoacid residues, and three positions were kept constant. Twooligonucleotides, generated by the TRIM technology, which enablesincorporation of randomized sets of trinucleotide building blocks, wereordered from Ella Biotech (Martinsried, Germany). The oligonucleotidesrepresented helix 1 and helix 2 of the Z molecule, respectively, andcontained an annealing region of 18 overlapping nucleotides. Theassembled double stranded DNA sequence was: 5′-AA ATA AAT CTC GAG GTAGAT GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN CTGCCT AAC CTC ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GACCCA AGC CAG AGC TCA TTA TTT A-3′ (SEQ ID NO:1680; designed codons aredenoted NNN). The sequence encodes a partially randomized helix 1 and 2of the Z variant amino acid sequence flanked by the restriction sitesXhoI and SacI. The design for each amino acid residue of the newlibrary, including ten variable amino acid positions (9, 10, 11, 13, 14,17, 18, 25, 32 and 35) and three constant amino acid positions (24, 27and 28) in the Z molecule scaffold, are displayed in Table 7. Theresulting theoretical library size was 9.7×10⁸ variants. An eventheoretical distribution of the different amino acids was applied withineach amino acid position, except from position 9, 13, 17, 18, 32 and 35,where a higher portion of selected amino acids was applied.

TABLE 7 Design of first maturation library Zlib006IL-1RI.I. Amino acidAmino acid randomization No. of position in the (percentage when unevendifferent Z variant molecule distribution) amino acids 9 A(30%), E, F,H, I, K, L, Q, R, S, 14 T, V, W, Y 10 A, D, E, F, H, I, K, L, Q, R, S,T, 15 V, W, Y 11 A, D, E, F, H, I, K, L, N, Q, R, S, 16 T, V, W, Y 13 A,F, H, I, L, Q(20%), S, T, V, W, 11 Y(20%) 14 A, D, E, F, H, I, K, L, M,Q, R, S, 16 T, V, W, Y 17 F, W(20%), Y 3 18 A(11%), D, E, F, G, H, I, K,L, N, 17 Q, R, S, T, V, W, Y 24 R 1 25 H, K, R, S 4 27 Y 1 28 T 1 32 A,I, L, R(70%) 4 35 F(80%), L(20%) 2

Library construction: The two oligonucleotides were assembled,amplified, cloned into vector pAY02592, and transformed into E. coliER2738, essentially as described for matured libraries in WO2015/189430. Clones from the Zlib0061L-1 RI.I library were sequenced (asdescribed in WO 2009/077175) in order to verify the content and toevaluate the outcome of the constructed library vis-à-vis the librarydesign.

Preparation of phaqe stock: A phage stock containing the phagemidlibrary was prepared in shake flasks in two batches. Cells from aglycerol stock containing the phagemid library were inoculated in 2 and3 l, respectively, of TSB-YE medium, supplemented with 2% glucose, 10μg/ml tetracycline and 100 μg/ml ampicillin. The cultivations were grownat 37° C. until OD₆₀₀ reached log-phase 0.5-0.9. The cultivations wereinfected using a 4-10×molar excess of M13K07 helper phage and wereincubated for 30 minutes at 37° C. Batch 1 cultivations were pelleted bycentrifugation, dissolved in TSB-YE medium, supplemented with 100 μg/mlampicillin, 25 μg/ml kanamycin and 0.1 mM IPTG and grown at 30° C.overnight. To batch 2 cultivation, 0.1 mM IPTG was added, followed by anincubation of 1 h at 37° C. After addition of 25 μg/ml kanamycin, thecultures were grown at 30° C. overnight. For both batches, the overnightgrown cells were pelleted by centrifugation at 4,000 g and the phageparticles remaining in the medium were thereafter precipitated twice inPEG/NaCl, filtered and dissolved in PBS and glycerol as described inGrönwall et al., supra. Phage stocks were stored at −80° C. until use inselection.

Results

Library construction: The new library Zlib0061L-1 RI.I was designedbased on a set of IL-1R-I binding Z variants with verified bindingproperties (Example 1 and 2). The theoretical size of the designedlibrary was 9.7×10⁸ Z variants. The actual size of the library,determined by titration after transformation to E. coli ER2738 cells,was 5.6×10⁹ transformants. The library quality was tested by sequencing192 transformants and by comparing their actual sequences with thetheoretical design. The contents of the actual library compared to thedesigned library were shown to be satisfactory. A first matured libraryof potential binders to IL-1R-I was thus successfully constructed.

Example 4 Selection and Screening of Z Variants from the First MaturedLibrary

In this Example, human and cynomolgus IL-1R-I were used as targetproteins in phage display selections using a matured phage library of Zvariants. The DNA of selected clones was sequenced, the Z variants wereproduced in E. coli periplasmic fractions and assayed against IL-1R-I inELISA and Biacore.

Materials and Methods

Expression of target protein: Expression of Fc fused cynomolgus IL-1R-I(cIL-1R-I-Fc, SEQ ID NO:1666) was performed using the Expi293 expressionsystem (Thermo Fisher Scientific), essentially according to themanufacturer's protocol. Supernatants were harvested by centrifugation 7days after transfection of expression vectors and stored at −70° C. Thefrozen supernatant from the Expi293 culture were thawed and filtrated(0.22 μm). The supernatant containing the cIL-1R-I-Fc was purified usingaffinity chromatography with a MabSelect SuRe column. The purifiedprotein was buffer exchanged to PBS. The purity of the protein wasanalyzed by SDS-PAGE stained with Coomassie Blue and the molecularweight was analyzed using mass spectrometry (HPLC/MS or MALDI-TOF/MS).

Phage display selection of IL-1R-1 binding Z variants: The biotinylatedhuman target proteins b-hIL-1R-I-Fc and b-hIL-1R-1, and the cynomolgustarget proteins cIL-1R-I-Fc (SEQ ID NO:1666) and b-cIL-1R-I-Fc(cIL-1RI-Fc biotinylated as hIL-1R-I-Fc in Example 1), were used inphage selections using the new library of Z variant molecules describedin Example 3. An overview of the selection strategy, describing theparallel selection tracks and an overall increased stringency in theselection cycles obtained by using a lowered target concentration and anincreased number of washes, is shown in Table 8. The selections wereperformed in four to five cycles essentially as described in Example 1,with the following exceptions 1-6. Exception 1: pre-selection wasperformed for >60 min at RT by incubation of phage stocks with Fc/SAbeads (selection tracks 1-1, 1-2, 1-4, 1-6, 1-8, 2-14 and 2-16), SAbeads (selection tracks 1-3, 1-7, 1-9, 2-15 and 2-17) or Fc/protein Abeads (selection track 1-5), respectively. Exception 2: the time forselection was 30-100 min as presented for each track in Table 8Exception 3: target-phage complexes were captured on beads using thefollowing amount of target protein and beads: 8 μg b-hIL-1R-I-Fc, 1 μgb-hIL-1R-I or 4 μg cIL-1R-I-Fc per mg SA beads, respectively, and 5 μgcIL-1R-I-Fc per mg protein A beads. In selection round 5, 2 mg SPHEROneutravidin beads (Spherotech cat. no. NVM-20-5) were used fortarget-phage complex capture. Exception 4: in the second final wash stepof tracks 2-14, 2-15, 2-17, 3-14, 3-15 and 3-17, respectively, hIL-1R-Iwas added to the wash buffer in 100 times higher concentration than thetarget concentration of the respective track and the wash was run for15-20 min. Exception 5: the phage/target/bead complexes of selectiontracks 4-1 to 4-13 were divided into two after the bead wash procedure.Half of the samples was submitted to elution and the other half, denoted4-1× to 4-13×, was submitted to an extra 66 h, wash step before elution.Exception 6: two selection rounds four tracks, 4-4× and 4-6×, weresubmitted to a fifth selection round, resulting in tracks 5-1 and 5-2,as presented in Table 8. E. coli XL-1 Blue cells (Agilent Technologies,cat. no. 200268), grown in medium supplemented with 10 μg/mltetracycline, were used for phage infection and a 10× excess of M13K07helper phage compared to bacteria was allowed to infect log phasebacteria. Amplification of phage particles after round 1 to 3 wasperformed essentially as described for round 2 and 3 in Example 1.

TABLE 8 Overview of selections from the first matured library. Phagestock Target Number Selection from library or concentration Selection ofCycle track selection track Target (nM) time (min) washes 1 1-1Zlib006IL-1RI.I b-hIL-1R-I-Fc 50 70 4 1 1-2 Zlib006IL-1RI.Ib-hIL-1R-I-Fc 10 70 4 1 1-3 Zlib006IL-1RI.I b-hIL-1R-I 50 70 4 1 1-4Zlib006IL-1RI.I b-cIL-1R-I-Fc 100 70 2 1 1-5 Zlib006IL-1RI.I cIL-1R-I-Fc100 70 2 1 1-6 Zlib006IL-1RI.I b-hIL-1R-I-Fc 10 45 5 1 1-7Zlib006IL-1RI.I b-hIL-1R-I 10 45 5 1 1-8 Zlib006IL-1RI.I b-hIL-1R-I-Fc10 45 5 1 1-9 Zlib006IL-1RI.I b-hIL-1R-I 10 45 5 2 2-1 1-1 b-hIL-1R-I-Fc20 60 6 2 2-2 1-1 b-hIL-1R-I-Fc 5 60 6 2 2-3 1-2 b-hIL-1R-I-Fc 4 60 8 22-4 1-2 b-hIL-1R-I-Fc 4 60 12  2 2-5 1-3 b-hIL-1R-I 20 60 6 2 2-6 1-3b-hIL-1R-I 10 60 6 2 2-7 1-3 b-hIL-1R-I 10 60 10  2 2-8 1-4b-cIL-1R-I-Fc 50 60 4 2 2-9 1-5 cIL-1R-I-Fc 50 60 4 2 2-10 1-1b-cIL-1R-I-Fc 100 60 2 2 2-11 1-1 b-cIL-1R-I-Fc 50 60 6 2 2-12 1-1cIL-1R-I-Fc 100 60 2 2 2-13 1-1 cIL-1R-I-Fc 50 60 6 2 2-14 1-8b-hIL-1R-I-Fc 2 30 10* 2 2-15 1-9 b-hIL-1R-I 2 30 10* 2 2-16 1-9b-cIL-1R-I-Fc 25 30 3 2 2-17 1-6/1-7 pool b-hIL-1R-I 2 30 10* 3 3-1 2-1b-hIL-1R-I-Fc 5 60 8 3 3-2 2-2 b-hIL-1R-I-Fc 0.5 60 8 3 3-3 2-3b-hIL-1R-I-Fc 1 60 12  3 3-4 2-4 b-hIL-1R-I-Fc 1 60 24  3 3-5 2-5b-hIL-1R-I 5 60 8 3 3-6 2-6 b-hIL-1R-I 1 60 8 3 3-7 2-7 b-hIL-1R-I 2 6020  3 3-8 2-8 b-cIL-1R-I-Fc 25 60 6 3 3-9 2-9 cIL-1R-I-Fc 25 60 6 3 3-102-10 b-hIL-1R-I-Fc 25 60 6 3 3-11 2-11 b-hIL-1R-I-Fc 5 60 8 3 3-12 2-12b-hIL-1R-I-Fc 25 60 6 3 3-13 2-13 b-hIL-1R-I-Fc 5 60 8 3 3-14 2-14b-hIL-1R-I-Fc 0.5 40 16* 3 3-15 2-15 b-hIL-1R-I 0.5 40 16* 3 3-16 2-16b-hIL-1R-I 2 40 9 3 3-17 2-17 b-hIL-1R-I 0.4 40 16* 4 4-1 3-1b-hIL-1R-I-Fc 1 40  10** 4 4-2 3-2 b-hIL-1R-I-Fc 0.05 40  10** 4 4-3 3-3b-hIL-1R-I-Fc 0.2 40  16** 4 4-4 3-4 b-hIL-1R-I-Fc 0.2 40  36** 4 4-53-5 b-hIL-1R-I 1 40  10** 4 4-6 3-6 b-hIL-1R-I 0.1 40  10** 4 4-7 3-7b-hIL-1R-I 0.4 40  30** 4 4-8 3-8 b-cIL-1R-I-Fc 12.5 40  8** 4 4-9 3-9cIL-1R-I-Fc 50 40  8** 4 4-10 3-10 b-cIL-1R-I-Fc 50 40  6** 4 4-11 3-11b-cIL-1R-I-Fc 10 40  12** 4 4-12 3-12 cIL-1R-I-Fc 50 40  6** 4 4-13 3-13cIL-1R-I-Fc 10 40  12** 4 4-14 3-14 b-hIL-1R-I-Fc 0.1 40 20  4 4-15 3-15b-hIL-1R-I 0.1 40 20  4 4-16 3-16 b-cIL-1R-I-Fc 5 40 16  4 4-17 3-17b-hIL-1R-I 0.2 40 16  5 5-1 4-4x b-hIL-1R-I-Fc 50 100 3 5 5-2 4-6xb-hIL-1R-I 50 100 3 *Addition of hlL-1R-I in the second final wash step.**Selection track divided into two after washes. One part submitted toan extra wash, over the weekend, before elution.

In the first selection cycle, nine selection tracks were run; 1-1 to1-9. In cycle two, some tracks were divided into two, three or sixtracks and two tracks were pooled into one track, resulting in a totalof 17 parallel tracks in cycle 2, 3 and 4; 2-1 to 2-17, 3-1 to 3-17 and4-1 to 4-17, respectively. In cycle 5, two tracks were run; 5-1 and 5-2.In selection tracks 1-1 to 1-5, batch 1 of Zlib0061L-1R1.I was used, andin 1-6 to 1-9, batch 2 was used, respectively. All selection tracks arepresented in Table 8. The number of phage particles used for selectionswas typically more than 2,000 times the number of eluted phage particlesin the previous cycle.

Sequencing: Individual clones from cycle 4 or 5 of the differentselection tracks were picked for sequencing. Amplification and sequenceanalysis of gene fragments were performed essentially as described inExample 1.

Production of Z variants for ELISA and Biacore screening: Z variantswere produced essentially as described in Example 1 with the exceptionthat the culture volume was 1.2 ml and that the periplasmic extractswere clarified by filtration using 1.2 μm 96 well filter plates(MerckMillipore cat. no. MSANLY50) after the freeze-thawing procedure. Zvariants screened in Biacore were diluted five times in HBS-EP buffer.

ELISA screening of Z variants: The binding of Z variants to human wasanalyzed in ELISA, essentially as described in Example 1, using 0.3 nMhIL-1R-1-Fc as target protein. As blank control, PBST 0.05% was addedinstead of the Z-ABD periplasmic extract.

EC₅₀ analysis of Z variants: A selection of IL-1R-I binding Z variantswas subjected to an analysis of response against a dilution series ofhIL-1R-1-Fc using ELISA as described above. The target proteinhIL-1R-1-Fc was diluted stepwise 1:10 from 100 to 0.01 nM. As abackground control, the Z variants were assayed with no target proteinadded. Obtained data was analyzed using GraphPad Prism 5 and non-linearregression, and the EC₅₀ values (the half maximal effectiveconcentration) were calculated. A periplasmic extract with Z12967 (SEQID NO:9) was analyzed in parallel for signal comparison.

Biacore screening of Z variants: The binding of Z variants to humanIL-1R-I was analyzed in a kinetic screening using a Biacore T200instrument. A polyclonal goat anti-ABD antibody (goat anti-ABD) wasimmobilized on CM5 chip surfaces basically as described for otherproteins in Example 2. For the kinetic screening, analytes were injectedin two steps. First, a Z-ABD (ABD001, SEQ ID NO:1660) periplasmicextract was injected over the surface at 5 μl/min for 1 min. As a secondstep, 100 nM hIL-1R-I was injected at 30 μl/min for 2 min, followed by 2min of dissociation in running buffer HBS-EP. Glycine-HCl pH 2.0 (cat.no. BR100355, GE Healthcare) was used for regeneration of the antibodysurfaces between the cycles. The temperature of the assay was 25° C.Before performing the kinetic analyses, the signal from 100 nM hIL-1R-Iinjected over a reference surface containing goat anti-ABD but no Z-ABDsample was subtracted from the sensorgram of Z-ABD binding to hIL-1R-I.Rough screening affinities (K_(D)) were calculated from the referencesubtracted 100 nM hIL-1R-I response using a 1:1 binding model of theBiaEvaluation software 4.1 (GE Healthcare). A periplasmic extract ofZ12967 was included in the screening analysis for comparison.Periplasmic extracts of four Z variants were also submitted to a singlecycle kinetics (SCK) assay, using the same setup as above, but injectingfive hIL-1R-I concentrations and evaluating the data with a 1:1 modelfor single cycle kinetics.

Results

Phage display selection of IL-1R-I binding Z variants: Individual cloneswere obtained after four or five cycles of phage display selectionsagainst human and cynomolgus IL-1R-I.

Sequencing: Sequencing was performed for clones obtained after four orfive cycles of selection. Each variant was given a unique identificationnumber #####, and individual variants are referred to as Z#####. Theamino acid sequences of the 58 amino acid residues long Z variants arelisted in FIG. 1 and in the sequence listing as SEQ ID NO:20-1209. Thededuced IL-1R-I binding motifs extend from residue 8 to residue 36 ineach sequence. The amino acid sequences of the 49 amino acid residueslong polypeptides predicted to constitute the complete three-helixbundle within each of these Z variants extend from residue 7 to residue55. It was observed that in one sequenced Z variant (Z18557, SEQ IDNO:1205) a valine residue was present in position 12.

ELISA screening of Z variants: Clones obtained after four or five cyclesof selection were produced individually in 96-well plates and werescreened for human IL-1R-I binding activity in ELISA. 98% of the assayedZ variants gave a positive signal of 2× the blank control or higheragainst 0.3 nM hIL-1R-I-Fc.

EC₅₀ analysis of Z variants: A subset of 115 Z variants displayingresults over 0.99 AU (16.5× the blank control) was subjected to ELISAtarget titration using hIL-1R-I-Fc. Obtained results were used forcalculation of EC₅₀ values (Table 9). The result for Z12967 (SEQ IDNO:9) was 2.4×10⁻¹⁰ M.

TABLE 9 Calculated EC₅₀ values from ELISA titration analysis. SEQ ID Zvariant NO EC₅₀ (M) Z15811 21 2.5 × 10⁻¹⁰ Z15813 23 3.5 × 10⁻¹⁰ Z1582434 2.3 × 10⁻¹⁰ Z15840 49 2.5 × 10⁻¹⁰ Z15862 71 2.1 × 10⁻¹⁰ Z15876 12064.2 × 10⁻¹⁰ Z15886 94 2.6 × 10⁻¹⁰ Z15897 105 2.5 × 10⁻¹⁰ Z15923 131 2.4× 10⁻¹⁰ Z15927 135 2.2 × 10⁻¹⁰ Z15929 137 2.4 × 10⁻¹⁰ Z15932 1207 2.3 ×10⁻¹⁰ Z15949 154 2.5 × 10⁻¹⁰ Z15953 158 2.1 × 10⁻¹⁰ Z15960 165 2.3 ×10⁻¹⁰ Z15961 166 2.1 × 10⁻¹⁰ Z15975 1209 2.4 × 10⁻¹⁰ Z15978 182 4.8 ×10⁻¹⁰ Z15987 191 2.3 × 10⁻¹⁰ Z15996 200 2.5 × 10⁻¹⁰ Z15997 201 2.5 ×10⁻¹⁰ Z15999 203 2.4 × 10⁻¹⁰ Z16000 204 2.4 × 10⁻¹⁰ Z16003 207 2.8 ×10⁻¹⁰ Z16004 208 2.5 × 10⁻¹⁰ Z16007 211 2.2 × 10⁻¹⁰ Z16008 212 2.3 ×10⁻¹⁰ Z16011 215 2.2 × 10⁻¹⁰ Z16023 227 2.1 × 10⁻¹⁰ Z16034 238 2.3 ×10⁻¹⁰ Z16041 245 2.4 × 10⁻¹⁰ Z16042 246 2.4 × 10⁻¹⁰ Z16043 247 2.3 ×10⁻¹⁰ Z16046 250 2.7 × 10⁻¹⁰ Z16048 252 2.5 × 10⁻¹⁰ Z16049 253 2.4 ×10⁻¹⁰ Z16050 254 2.3 × 10⁻¹⁰ Z16051 255 2.5 × 10⁻¹⁰ Z16053 257 2.5 ×10⁻¹⁰ Z16054 258 2.4 × 10⁻¹⁰ Z16058 262 2.3 × 10⁻¹⁰ Z16070 273 2.3 ×10⁻¹⁰ Z16072 275 2.2 × 10⁻¹⁰ Z16084 287 2.2 × 10⁻¹⁰ Z16089 291 2.4 ×10⁻¹⁰ Z16092 293 2.4 × 10⁻¹⁰ Z16093 294 2.1 × 10⁻¹⁰ Z16097 298 2.0 ×10⁻¹⁰ Z16102 302 2.8 × 10⁻¹⁰ Z16106 306 2.3 × 10⁻¹⁰ Z16116 316 2.1 ×10⁻¹⁰ Z16117 317 2.1 × 10⁻¹⁰ Z16121 321 2.3 × 10⁻¹⁰ Z16122 322 2.9 ×10⁻¹⁰ Z16127 325 2.1 × 10⁻¹⁰ Z16135 333 2.5 × 10⁻¹⁰ Z16141 339 2.8 ×10⁻¹⁰ Z16143 341 2.1 × 10⁻¹⁰ Z16155 351 2.9 × 10⁻¹⁰ Z16156 352 2.4 ×10⁻¹⁰ Z16161 357 2.5 × 10⁻¹⁰ Z16162 358 2.7 × 10⁻¹⁰ Z16163 359 1.7 ×10⁻¹⁰ Z16164 360 6.8 × 10⁻¹⁰ Z16165 361 2.2 × 10⁻¹⁰ Z16168 364 2.8 ×10⁻¹⁰ Z16169 365 1.8 × 10⁻¹⁰ Z16173 369 3.6 × 10⁻¹⁰ Z16178 374 2.4 ×10⁻¹⁰ Z16206 402 4.5 × 10⁻¹⁰ Z16207 403 2.2 × 10⁻¹⁰ Z16219 415 2.4 ×10⁻¹⁰ Z16234 429 5.4 × 10⁻¹⁰ Z16236 431 2.4 × 10⁻¹⁰ Z16241 436 4.9 ×10⁻¹⁰ Z16243 438 2.2 × 10⁻¹⁰ Z16245 440 2.2 × 10⁻¹⁰ Z16246 441 3.9 ×10⁻¹⁰ Z16255 449 1.7 × 10⁻¹⁰ Z16263 457 2.6 × 10⁻¹⁰ Z16264 458 3.0 ×10⁻¹⁰ Z16277 470 2.6 × 10⁻¹⁰ Z16301 494 2.2 × 10⁻¹⁰ Z16302 495 2.8 ×10⁻¹⁰ Z16305 498 2.2 × 10⁻¹⁰ Z16312 504 2.4 × 10⁻¹⁰ Z16319 511 3.0 ×10⁻¹⁰ Z16321 513 2.7 × 10⁻¹⁰ Z16329 519 4.5 × 10⁻¹⁰ Z16341 531 1.7 ×10⁻¹⁰ Z16363 550 2.0 × 10⁻¹⁰ Z16377 562 2.1 × 10⁻¹⁰ Z16378 563 2.0 ×10⁻¹⁰ Z16388 573 2.9 × 10⁻¹⁰ Z16405 590 3.6 × 10⁻¹⁰ Z16678 853 2.4 ×10⁻¹⁰ Z16681 856 2.3 × 10⁻¹⁰ Z16688 863 2.7 × 10⁻¹⁰ Z16749 921 2.7 ×10⁻¹⁰ Z16771 943 2.4 × 10⁻¹⁰ Z17244 1147 4.2 × 10⁻¹⁰ Z17255 1158 2.5 ×10⁻¹⁰ Z17257 1160 4.8 × 10⁻¹⁰ Z17269 1171 2.7 × 10⁻¹⁰ Z17270 1172 3.0 ×10⁻¹⁰ Z17274 1176 2.1 × 10⁻¹⁰ Z17278 1180 2.7 × 10⁻¹⁰ Z17279 1181 2.8 ×10⁻¹⁰ Z17280 1182 2.2 × 10⁻¹⁰ Z17288 1190 4.2 × 10⁻¹⁰ Z17292 1194 2.0 ×10⁻¹⁰ Z17296 1198 3.2 × 10⁻¹⁰ Z17297 1199 2.4 × 10⁻¹⁰ Z18557 1205 1.9 ×10⁻¹⁰

On average, the Z variants originating from this library (first maturedlibrary) displayed an improvement in EC₅₀ values by a factor 10 comparedwith the average EC₅₀ for the Z variants originating from the primaryselection.

Biacore screening of Z variants: A selection of IL-1R-I binding Zvariants was submitted to a Biacore kinetic screening. A singleconcentration of hIL-1R-I was injected over each Z-ABD captured fromperiplasmic extracts on a sensor chip surface containing an anti-ABDantibody. The calculated screening affinities are presented in Table 10.Z16062 (SEQ ID NO:266) got the best K value (7.6×10⁻⁹ M) of the assayedbinders. The K_(D) of Z2967 was 2.5×10⁻⁸ M. The four binders assayed ina S1K experiment got K_(D) values that deviated from their respectivescreening kinetics affinities by 0-14%.

TABLE 10 Calculated K_(D) values from Biacore kinetic screening. SEQ IDZ variant NO K_(D) (M) Z15824 34 9.2 × 10⁻⁹ Z15840 49 1.1 × 10⁻⁸ Z1586473 1.6 × 10⁻⁸ Z15873 82 2.3 × 10⁻⁸ Z15876 1206 8.3 × 10⁻⁹ Z15882 90 9.0× 10⁻⁹ Z15889 97 1.5 × 10⁻⁸ Z15897 105 1.2 × 10⁻⁸ Z15908 116 1.7 × 10⁻⁸Z15947 152 1.1 × 10⁻⁸ Z15953 158 1.1 × 10⁻⁸ Z15972 177 1.2 × 10⁻⁸ Z15986190 9.5 × 10⁻⁹ Z16009 213 2.0 × 10⁻⁸ Z16010 214 1.2 × 10⁻⁸ Z16011 2151.1 × 10⁻⁸ Z16012 216 1.2 × 10⁻⁸ Z16013 217 1.8 × 10⁻⁸ Z16014 218 1.1 ×10⁻⁸ Z16015 219 1.5 × 10⁻⁸ Z16016 220 1.2 × 10⁻⁸ Z16017 221 1.8 × 10⁻⁸Z16018 220 1.6 × 10⁻⁸ Z16019 223 1.5 × 10⁻⁸ Z16020 224 2.0 × 10⁻⁸ Z16021225 9.6 × 10⁻⁹ Z16022 226 1.5 × 10⁻⁸ Z16023 227 9.2 × 10⁻⁹ Z16024 2281.4 × 10⁻⁸ Z16025 229 1.9 × 10⁻⁸ Z16026 230 1.6 × 10⁻⁸ Z16027 231 1.4 ×10⁻⁸ Z16028 232 1.9 × 10⁻⁸ Z16029 233 2.1 × 10⁻⁸ Z16030 234 1.7 × 10⁻⁸Z16031 235 2.0 × 10⁻⁸ Z16032 236 2.6 × 10⁻⁸ Z16033 237 1.9 × 10⁻⁸ Z16034238 9.1 × 10⁻⁹ Z16035 239 1.7 × 10⁻⁸ Z16036 240 1.1 × 10⁻⁸ Z16037 2411.4 × 10⁻⁸ Z16038 242 8.9 × 10⁻⁹ Z16039 243 1.4 × 10⁻⁸ Z16040 244 2.0 ×10⁻⁸ Z16041 245 1.0 × 10⁻⁸ Z16042 246 9.7 × 10⁻⁹ Z16043 247 8.3 × 10⁻⁹Z16044 248 1.7 × 10⁻⁸ Z16045 249 2.0 × 10⁻⁸ Z16046 250 1.8 × 10⁻⁸ Z16047251 1.9 × 10⁻⁸ Z16048 252 1.1 × 10⁻⁸ Z16049 253 1.4 × 10⁻⁸ Z16050 2541.3 × 10⁻⁸ Z16051 255 1.5 × 10⁻⁸ Z16052 256 1.2 × 10⁻⁸ Z16053 257 1.1 ×10⁻⁸ Z16054 258 2.9 × 10⁻⁸ Z16055 259 1.2 × 10⁻⁸ Z16056 260 1.3 × 10⁻⁸Z16057 261 3.3 × 10⁻⁸ Z16058 262 1.4 × 10⁻⁸ Z16059 263 1.6 × 10⁻⁸ Z16060264 1.9 × 10⁻⁸ Z16061 265 1.2 × 10⁻⁸ Z16062 266 7.6 × 10⁻⁹ Z16063 2671.2 × 10⁻⁸ Z16065 268 7.8 × 10⁻⁹

Example 5 Production and Characterization of IL-1R-I Binding Z Variantsfrom the First Matured Library

This Example describes the general procedure for subcloning andproduction of His₆-tagged and ABD-fused Z variants (originating from thefirst maturation library phage selection), characterization of theirIL-1R-I binding, blocking and melting points.

Materials and Methods

Subcloning of Z variants with a His₆ tag: The DNA of the respective Zvariant was amplified from the phage library vector pAY02592 and wassubcloned with an N-terminal His₆ tag using standard molecular biologytechniques essentially as described in Example 2.

Subcloning of Z variants in fusion with ABD: The N terminal sequence ofrespective Z variant was mutated, in position 1 and 2 to amino acidresidues A and E respectively, using standard molecular biologytechniques. The resulting new Z variants were subcloned into anexpression vector containing an ABD variant, giving the encodingsequence [Z#####]-ASGS-ABD, where the ABD variant was PP013 (SEQ IDNO:1661). Z##### refers to individual, 58 amino acid residue long Zvariants.

Cultivation: E. coli T7E2 cells (GeneBridges) were transformed withplasmids containing the gene fragment of each respective IL-1R-I bindingZ variant. The resulting recombinant strains were cultivated in mediasupplemented with 50 μg/ml kanamycin at 30° C. in 50 ml scale using theEnPresso protocol (BioSilta). In order to induce protein expression,IPTG was added to a final concentration of 0.2 mM at OD600≈10. Afterinduction, the cultivations were incubated for 16 h. The cells wereharvested by centrifugation.

Purification of IL-1R-I binding Z variants with a His₆ tag: Purificationwas performed essentially as described in Example 2, but without thebuffer exchange between the IMAC and RPC purification steps for themajority of the samples. During RPC purification, the RPC solvent A waschanged to 0.1% TFA in 100% water and the linear gradient was 0-60% RPCsolvent B for 18 ml.

Purification of IL-1R-I binding Z variants with ABD fusion:Approximately 2 g of each cell pellet was re-suspended in TST-buffer (25mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05% Tween20, pH 8.0) supplementedwith Benzonase® (Merck). After cell disruption and clarification bycentrifugation, each supernatant was applied on a gravity flow columnwith 1 ml agarose immobilized with an anti-ABD ligand (producedin-house). After washing with TST-buffer and 5 mM NH₄Ac pH 5.5 buffer,the ABD fused Z variants were eluted with 0.1 M HAc. The buffer of theeluate was exchanged to PBS (2.68 mM KCl, 137 mM NaCl, 1.47 mM KH₂PO₄,8.1 mM Na₂HPO₄, pH 7.4) using PD-10 desalting columns (GE Healthcare).

Protein concentrations were determined by measuring the absorbance at280 nm, using a NanoDrop® ND-1000 spectrophotometer (Saveen Werner AB)and the extinction coefficient of the respective protein. The purity wasanalyzed by SDS-PAGE stained with Coomassie Blue and the identity ofeach purified Z variant was confirmed using LC/MS analysis.

Biacore kinetic analysis: The kinetic constants (k_(on) and k_(off)) andaffinities (K_(D)) for human and cynomolgus IL-1R-I were determined forHis₆-tagged Z variants using a Biacore 2000 instrument (GE Healthcare).The experiment was run essentially as described in Example 2, using 100,10 and 1 nM of the Z variants. The analyte injection time was 3 minfollowed by 15 min dissociation and 2×5 s glycine pH 3.0 (GE Healthcare,cat. no. BR100357), supplemented with 0.5 M NaCl, was used forregeneration of the surfaces between the cycles. Kinetic constants werecalculated from the obtained sensorgrams of two or three concentrationsof the respective Z variant, using a 1:1 binding model in theBiaEvaluation software 4.1 (GE Healthcare). His₆-tagged Z12967 wasincluded in the assay for comparison.

In vitro IL-1β neutralization assay: His₆-tagged IL-1R-I specific Zvariants were tested for their inhibitory capacity in the TF-1 cellassay. The assay was run as described in Example 2. The data on cellgrowth was assessed by non-linear regression to a four-parameterdose-response curve, and IC₅₀ values were determined using GraphPadPrismprogram. His₆-tagged Z12967 was included for comparison.

Circular dichroism (CD) spectroscopy analysis: CD was analyzed forHis₆-tagged Z variants, as described in Example 2.

Results

Subcloning with His₆ tag: Z variants were subcloned into constructs withan N-terminal His₆.

Production of His₆-tagged Z variants: The IL-1R-I binding Z variantswith a His₆ tag were expressed as soluble gene products in E. coli. Theamount of purified protein was determined spectrophotometrically bymeasuring the absorbance at 280 nm and ranged from approximately 1 to 7mg protein per g pellet. SDS-PAGE analysis of each final proteinpreparation showed that these predominantly contained the IL-1R-Ibinding Z variant. The correct identity and molecular weight of each Zvariant were confirmed by HPLC-MS analysis.

Biacore kinetic analysis: The interactions of His₆-taggedIL-1R-1-binding Z variants with human and cynomolgus IL-1R-I wereanalyzed in a Biacore instrument by injecting various concentrations ofthe Z variants over a surface containing immobilized IL-1R-1. A summaryof the calculated kinetic parameters (K_(D), k_(on) and k_(off)) for thehuman IL-1R-I binding is given in Table 11. The strongest binder tohuman IL-1R-1, Z18557 (SEQ ID NO:1205), got a K_(D) of 1.5×10⁻¹⁰ againsthuman IL-1R-I and 7.6×10⁻⁹ M against cynomolgus IL-1R-1. The K_(D) ofZ12967 to human IL-1R-I was 5.1×10⁻¹⁰ M.

TABLE 11 Kinetic parameters and affinities for His₆-Z polypeptidesbinding to human IL-1R-I. Z variant SEQ ID NO k_(on) (1/Ms) k_(off)(1/s) K_(D) (M) Z15862 71 2.8 × 10⁶ 1.9 × 10⁻³ 6.7 × 10⁻¹⁰ Z15876 12062.1 × 10⁶ 1.3 × 10⁻³ 6.2 × 10⁻¹⁰ Z15927 135 2.4 × 10⁶ 1.2 × 10⁻³ 5.1 ×10⁻¹⁰ Z15953 158 6.0 × 10⁶ 1.5 × 10⁻³ 2.4 × 10⁻¹⁰ Z15961 166 4.3 × 10⁶2.0 × 10⁻³ 4.6 × 10⁻¹⁰ Z16007 211 2.3 × 10⁶ 2.1 × 10⁻³ 9.2 × 10⁻¹⁰Z16023 227 5.3 × 10⁶ 1.5 × 10⁻³ 2.9 × 10⁻¹⁰ Z16043 247 2.5 × 10⁶ 1.1 ×10⁻³ 4.4 × 10⁻¹⁰ Z16062 266 2.6 × 10⁶ 1.1 × 10⁻³ 4.4 × 10⁻¹⁰ Z16065 2686.9 × 10⁶ 1.3 × 10⁻³ 1.9 × 10⁻¹⁰ Z16072 275 4.6 × 10⁶ 2.2 × 10⁻³ 4.7 ×10⁻¹⁰ Z16093 294 5.6 × 10⁶ 1.4 × 10⁻³ 2.5 × 10⁻¹⁰ Z16097 298 3.1 × 10⁶1.5 × 10⁻³ 4.8 × 10⁻¹⁰ Z16116 316 2.6 × 10⁶ 2.3 × 10⁻³ 8.8 × 10⁻¹⁰Z16117 317 4.0 × 10⁶ 2.7 × 10⁻³ 6.9 × 10⁻¹⁰ Z16127 325 6.7 × 10⁶ 1.9 ×10⁻³ 2.9 × 10⁻¹⁰ Z16143 341 3.1 × 10⁶ 1.6 × 10⁻³ 5.3 × 10⁻¹⁰ Z16163 3593.5 × 10⁶ 2.0 × 10⁻³ 5.5 × 10⁻¹⁰ Z16165 361 2.1 × 10⁶ 3.6 × 10⁻³ 1.7 ×10⁻⁹  Z16169 365 2.6 × 10⁶ 1.0 × 10⁻³ 3.9 × 10⁻¹⁰ Z16219 415 2.5 × 10⁶5.6 × 10⁻³ 2.2 × 10⁻⁹  Z16245 440 2.7 × 10⁶ 3.1 × 10⁻³ 1.2 × 10⁻⁹ Z16255 449 3.0 × 10⁶ 3.1 × 10⁻³ 1.0 × 10⁻⁹  Z16301 494 3.1 × 10⁶ 2.1 ×10⁻³ 6.6 × 10⁻¹⁰ Z16341 531 3.0 × 10⁶ 1.6 × 10⁻³ 5.2 × 10⁻¹⁰ Z16363 5503.2 × 10⁶ 1.8 × 10⁻³ 5.7 × 10⁻¹⁰ Z16377 562 2.1 × 10⁶ 1.1 × 10⁻³ 5.4 ×10⁻¹⁰ Z16378 563 6.6 × 10⁶ 3.6 × 10⁻³ 5.5 × 10⁻¹⁰ Z16681 856 3.6 × 10⁶5.0 × 10⁻³ 1.4 × 10⁻⁹  Z17274 1176 1.1 × 10⁷ 2.4 × 10⁻³ 2.2 × 10⁻¹⁰Z17280 1182 2.4 × 10⁶ 5.3 × 10⁻³ 2.2 × 10⁻⁹  Z17292 1194 1.3 × 10⁷ 5.0 ×10⁻³ 3.8 × 10⁻¹⁰ Z18557 1205 4.4 × 10⁶ 6.7 × 10⁻⁴ 1.5 × 10⁻¹⁰

In vitro IL-1β neutralization assay: The IL-1β inhibition ability ofHis₆-tagged IL-1R-1-binding Z variants was analyzed in a TF-1 cellassay. The resulting IC₅₀ values are presented in Table 12. Z18557 (SEQID NO:1205) displayed the best inhibition capacity with an IC₅₀ valuebelow 0.5 nM. The IC₅₀ of Z12967 was 5.2 nM.

TABLE 12 IC₅₀ values for His₆-Z polypeptides from a TF-1 cell assay. Zvariant SEQ ID NO IC₅₀ (nM) Z15862 71 6.6 Z15876 1206 2.4 Z15927 135 2.5Z15953 158 3.7 Z15961 166 2.9 Z16007 211 5.9 Z16023 227 3.8 Z16043 2472.5 Z16062 266 1.8 Z16065 268 1.7 Z16072 275 4.3 Z16093 294 3.3 Z16097298 3.4 Z16116 316 4.1 Z16117 317 8.5 Z16127 325 4.1 Z16143 341 4.8Z16163 359 1.8 Z16165 361 11.5 Z16169 365 0.76 Z16219 415 24.2 Z16245440 8.2 Z16255 449 8.5 Z16301 494 4.5 Z16341 531 3.9 Z16363 550 8.1Z16377 562 3.1 Z16378 586 32.7 Z16681 856 20.1 Z17274 1176 4.6 Z172801182 5.4 Z17292 1194 13.2 Z18557 1205 <0.5

CD analysis: The CD spectra determined for the IL-1R-I binding Zvariants with a His₆ tag showed that each had an a-helical structure at20° C. This result was also verified in the variable temperaturemeasurements, wherein melting temperatures were determined (Table 13).Reversible folding was seen for all the IL-1R-I binding Z variants whenoverlaying spectra measured before and after heating to 90° C.

TABLE 13 Melting temperatures (Tm). Z variant SEQ ID NO Tm (° C.) Z158761206 55 Z15927 135 56 Z15953 158 56 Z15961 166 59 Z16023 227 60 Z16043247 54 Z16062 266 54 Z16065 268 59 Z16093 294 52 Z16097 298 56 Z16116316 59 Z16117 317 63 Z16127 325 59 Z16163 359 57 Z16169 365 48 Z16245440 63 Z16341 531 59 Z16377 562 52 Z16681 856 68 Z17280 1182 62 Z185571205 60

Example 6 Design and Construction of Two Second Matured Libraries ofIL-1R-I Binding Z Variants

In this Example, two second matured libraries were constructed. Thelibraries were used for selections of additional IL-1R-I binding Zvariants.

Materials and Methods

Library design: The libraries were primarily based on sequences of humanIL-1R-I binding Z variants described in Example 4 and 5. In the newlibraries, denoted Zlib0061L-1 RI.II and Zlib0061L-1 RI.III, 10 variablepositions in the Z molecule scaffold were biased towards certain aminoacid residues and three positions were kept constant, respectively,according to a strategy mainly based on Z variants (SEQ ID NO:19-1209)from the selection from the first matured library. In addition, in bothlibraries, a new amino acid position was included for variation;position 12 of the 58 aa Z variant sequence, resulting in 11 variablepositions, in total. Position 12 of Z18557 (SEQ ID NO:1205),characterized in Example 5, was valine. The library design allowed forthe amino acids A, V and I in position 12.

For Zlib0061L-1 RI.II, a DNA linker was generated using split-poolsynthesis containing the following sequence ordered from DNA 2.0 (MenloPark, Calif., USA): 5′-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAAGAA NNN NNN NNN NNN NNN NNN GAG ATC NNN NNN CTG CCT AAC CTC ACC NNN NNNCAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTATTT A-3′ (SEQ ID NO:1681, designed codons are denoted NNN). The designfor each amino acid residue of the new library, including elevenvariable amino acid positions (9, 10, 11, 12, 13, 14, 17, 18, 25, 28 and35) and three constant amino acid positions (24, 27 and 32) in the Zmolecule scaffold, are displayed in Table 14. The resulting theoreticallibrary size was 2.2×10⁷ variants. An even theoretical distribution ofthe different amino acids was applied within each amino acid position,except from position 10, 11, 12, 13, 17, 28 and 35, where a higherportion of selected amino acids was applied.

For Zlib0061L-1 RI.III, the library was constructed similarly asZlib0061L-1 RI.I, described in Example 3, using the TRIM technology. TheDNA sequence, 5′-AA ATA AAT CTC GAG GTA GAT GCC AAA TAC GCC AAA GAA NNNNNN NNN NNN NNN NNN GAG ATC NNN NNN CTG CCT AAC CTC ACC NNN NNN CAA NNNNNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCA TTA TTT A-3(SEQ ID NO:1682, designed codons are denoted NNN) was ordered from EllaBiotech (Martinsried, Germany). The design for each amino acid residueof the new library, including eleven variable amino acid positions (9,10, 11, 12, 13, 14, 17, 18, 25, 32 and 35) and three constant amino acidpositions (24, 27 and 28) in the Z molecule scaffold, are displayed inTable 15. The resulting theoretical library size was 1.0×10⁸ variants.An even theoretical distribution of the different amino acids wasapplied within each amino acid position, except from position 10, 11,12, 32 and 35, where a higher portion of selected amino acids wasapplied.

TABLE 14 Zlib006IL-1RI.II library design, second maturation. Amino acidAmino acid randomization No. of position in the (percentage when unevendifferent Z variant molecule distribution) amino acids 9 A, E, I, L, T,V 6 10 E(50%), I, L, R, V, Y 6 11 A, E(27%), I, K, Q, R, T, V, Y 9 12A(50%), I, V 3 13 A, I, L, Q(50%), V, Y 6 14 F, M, Q, W, Y 5 17 F(70%),Y(30%) 2 18 A, D, E, F, G, H, I, K, L, Q, R, 16 S, T, V, W, Y 24 R 1 25K, R 2 27 Y 1 28 I, T(71.4%), V 3 32 R 1 35 F(25%), I(12.5%), L(50%), 4Y(12.5%)

TABLE 15 Zlib006IL-1RI.III library design, second maturation. Amino acidAmino acid randomization No. of position in the (percentage when unevendifferent Z variant molecule distribution) amino acids 9 A, E, I, L, T,V 6 10 E(60%), I, V 3 11 A, D, E(15%), F, H, I, K, L, Q, 15 R, S, T, V,W, Y 12 A(50%), I(20%), V(30%) 3 13 A, E, F, H, I, K, L, Q, R, S, 14 T,V, W, Y 14 A, E, F, H, I, K, L, Q, R, S, 14 T, V, W, Y 17 F, I, L, W, Y5 18 A, D, E, F, G, H, I, K, L, Q, 16 R, S, T, V, W, Y 24 R 1 25 K, R 227 Y 1 28 T 1 32 I(10%), R(90%) 2 35 F(30%), L(70%) 2

Library construction and phage stock preparation: Zlib0061L-1 RI.II andZlib0061L-1RI.III were constructed as described in WO 2016/113246(Example 4) and in Example 3, respectively. For both libraries, E. coliXL-1 Blue was used for library transformation. Clones from the librariesof Z variants were sequenced (as described in Example 3) in order toverify the content and to evaluate the outcome of the constructedlibrary vis-à-vis the library design.

Phage stocks containing the phagemid libraries were prepared in shakeflasks. Cells from glycerol stocks containing the phagemid library wereinoculated in 0.5 or 1 l, respectively, of TSB-YE medium, essentially asdescribed for Zlib0061L-1 RI.I batch 2 in Example 3. The cultivationswere infected using a 10-20× molar excess of M13K07 helper phage at anOD₆₀₀ of 0.8-0.9. Phage stocks were prepared as described in Example 3.

Results

Library construction: The new libraries were designed based on a set ofIL-1R-I binding Z variants, selected from Zlib0061L-1 RI.I, withverified binding properties (Examples 4 and 5). The theoretical sizes ofZlib0061L-1 RI.II and Zlib0061L-1 RI.III were 2.2×10⁷ and 1.0×10⁸ Zvariants, respectively. The actual sizes of the libraries, determined bytitration after transformation to E. coli XL-1 Blue cells, were 1.2×10⁹and 7.0×10⁹ transformants, respectively.

The library qualities were tested by sequencing of 192 and 96transformants from Zlib0061L-1 RI.II and Zlib0061L-1 RI.III,respectively, and by comparing their actual sequences with thetheoretical design. The contents of the actual library compared to thedesigned library were shown to be satisfactory. Two matured libraries ofpotential binders to IL-1R-I were thus successfully constructed.

Example 7 Selection of Z Variants from the Second Matured Libraries

In this Example, human and cynomolgus IL-1R-I were used as targetproteins in phage display selections using two second matured phagelibraries of Z variants.

Materials and Methods

Phage display selection of IL-1R-I binding Z variants: The biotinylatedhuman target proteins b-hIL-1R-I-Fc and b-hIL-1R-I, and the cynomolgustarget protein b-IL-1R-I-Fc, were used in phage selections using the newlibraries of Z variant molecules described in Example 6. An overview ofthe selection strategy, describing the parallel selection tracks and anoverall increased stringency in the selection cycles obtained by using alowered target concentration and an increased number of washes, is shownin Table 16.

TABLE 16 Overview of selections from the two second matured libraries.Phage stock from Target Selection library or selection concentrationSelection Number of Cycle track track Target (nM) time (min) washes 11-1 Zlib006IL-1RI.II b-hIL-1R-I-Fc 10 45  5 1 1-2 Zlib006IL-1RI.IIb-hIL-1R-I 10 45  5 1 1-3 Zlib006IL-1RI.II b-cIL-1R-I-Fc 25 45  3 1 1-4Zlib006IL-1RI.III b-hIL-1R-I-Fc 20 45  5 1 1-5 Zlib006IL-1RI.IIIb-hIL-1R-I 20 45  5 1 1-6 Zlib006IL-1RI.III b-cIL-1R-I-Fc 50 45  3 1 1-7Zlib006IL-1RI.II b-hIL-1R-I-Fc 10 45  5 1 1-8 Zlib006IL-1RI.IIb-hIL-1R-I 10 45  5 1 1-9 Zlib006IL-1RI.II b-cIL-1R-I-Fc 25 45  3 1 1-10Zlib006IL-1RI.III b-hIL-1R-I-Fc 20 45  5 1 1-11 Zlib006IL-1RI.IIIb-hIL-1R-I 20 45  5 1 1-12 Zlib006IL-1RI.III b-cIL-1R-I-Fc 50 45  3 22-1 1-1 b-hIL-1R-I-Fc 1 30  10* 2 2-2 1-2 b-hIL-1R-I 2 30 10 2 2-3 1-2b-hIL-1R-I 1 30 10 2 2-4 1-2 b-hIL-1R-I 1 30  10* 2 2-5 1-3 b-hIL-1R-I10 30  8 2 2-6 1-4 b-hIL-1R-I-Fc 4 30  8 2 2-7 1-5 b-hIL-1R-I 4 30  8 22-8 1-5 b-hIL-1R-I 2 30 10 2 2-9 1-5 b-hIL-1R-I 2 30  10* 2 2-10 1-6b-hIL-1R-I 20 30  6 2 2-11 1-7/1-8/1-9 pool b-hIL-1R-I 1 30  10* 2 2-121-10/1-11/1-12 pool b-hIL-1R-I 2 30  10* 3 3-1 2-1 b-hIL-1R-I-Fc 0.4 40 16* 3 3-2 2-2 b-hIL-1R-I 0.4 40 15 3 3-3 2-3 b-hIL-1R-I 0.1 40 20 3 3-42-4 b-hIL-1R-I 0.1 40  16* 3 3-5 2-5 b-cIL-1R-I-Fc 5 40 12 3 3-6 2-6b-hIL-1R-I-Fc 0.8 40 12 3 3-7 2-7 b-hIL-1R-I 0.8 40 12 3 3-8 2-8b-hIL-1R-I 0.2 40 20 3 3-9 2-9 b-hIL-1R-I 0.2 40  16* 3 3-10 2-10b-cIL-1R-I-Fc 10 40  9 3 3-11 2-11 b-hIL-1R-I 0.4 40  16* 3 3-12 2-12b-hIL-1R-I 0.4 40  16* 4 4-1 3-1 b-hIL-1R-I-Fc 0.125 40  20** 4 4-2 3-2b-hIL-1R-I 0.125 40 20 4 4-3 3-3 b-hIL-1R-I 0.05 40 30 4 4-4 3-4b-hIL-1R-I 0.05 40  20** 4 4-5 3-5 b-hIL-1R-I 1 40 16 4 4-6 3-6b-hIL-1R-I-Fc 0.16 40  16* 4 4-7 3-7 b-hIL-1R-I 0.16 40 16 4 4-8 3-8b-hIL-1R-I 0.1 40 30 4 4-9 3-9 b-hIL-1R-I 0.1 40  20** 4 4-10 3-10b-hIL-1R-I 2 40 16 4 4-11 3-11 b-hIL-1R-I 0.2 40 16 4 4-12 3-12b-hIL-1R-I 0.2 40 16 *Addition of hIL-1R-I in the second final washstep. **Selection track divided into two before second final wash step.One part submitted to an over-night wash in the presence of hIL-1R-I andone part submitted to a 1 h wash in PBST.

The selections were performed in four cycles essentially as described inExample 4, with the following exceptions 1-3. Exception 1: pre-selectionwas performed in selection round 1 and 2 for >60 min at RT by incubationof phage stocks with SA beads (b-hIL-1R-I tracks) or Fc/SA beads (b-IL-iRI-Fc tracks). Exception 2: in the second final wash step of tracks 2-i,2-4, 2-9, 2-11, 2-12, 3-i, 3-4, 3-9, 3-i11 and 3-12, respectively,hIL-1R-I was added to the wash buffer in 100 times higher concentrationthan the target concentration of the respective track and the wash wasrun for 15-20 min. Exception 3: In the fourth selection round, thetracks 4-i, 4-4 and 4-6 were divided into two before the second finalwash step. For one part, hIL-1R-I was added to the wash buffer in 100times higher concentration than the target concentration of therespective track and the second final wash was run overnight. For theother part, a 1 h wash in PBST was applied for the second final wash.

In the first selection cycle, twelve selection tracks were run; 1-1 to1-12. In cycle two, two tracks were divided into three, and six trackswere pooled into two tracks, resulting in a total of 12 parallel tracksin cycle 2, 3 and 4; 2-1 to 2-12, 3-1 to 3-12 and 4-1 to 4-12,respectively. All selection tracks are presented in Table 16. The numberof phage particles used for selections was typically more than 2000times the number of eluted phage particles in the previous cycle.

Results

Phage display selection of IL-1R-I binding Z variants: Individual cloneswere obtained after four cycles of phage display selections againsthuman and cynomolgus IL-1R-1.

Example 8 Screening of Z Variants from the First and Second MaturedLibraries

In this Example, DNA of selected clones from Example 4 and Example 7 wassequenced, the Z variants were produced in E. coli periplasmic fractionsand assayed against IL-1R-I in ELISA and Biacore.

Materials and Methods

Sequencing of potential binders: Individual clones from cycle 4 ofselections using the three matured libraries Zlib0061L-1 RI.I,Zlib0061L-1 RI.II and Zlib0061L-1 RI.III were picked for sequencing.Amplification and sequence analysis of gene fragments were performedessentially as described in Example 1.

Production of Z variants for ELISA and Biacore screening: Z variantswere produced essentially as described in Example 4 with the exceptionthat the periplasmic extracts were prepared by heat-treatment (82° C.,20 min) followed by clarification by filtration. Z variants screened inBiacore were diluted five times in HBS-EP buffer.

ELISA screening of Z variants: The binding of Z variants to humanIL-1R-I was analyzed in ELISA, essentially as described in Example 1,using 0.2 nM hIL-1R-1-Fc as target protein. As negative control, aperiplasmic extract of ABD001 was used.

EC₅₀ analysis of Z variants: A selection of IL-1R-I binding wassubjected to an analysis of response against a dilution series ofhIL-1R-1-Fc using ELISA as described above. The target proteinhIL-1R-1-Fc was diluted stepwise 1:10 from 20 to 0.002 nM. As abackground control, the Z variants were assayed with no target proteinadded. Obtained data was analyzed using GraphPad Prism 5 and non-linearregression, and the EC₅₀ values (the half maximal effectiveconcentration) were calculated. Periplasmic extracts with Z12967, Z16065and Z16218 (corresponding to SEQ ID:9, 268 and 414, respectively) wereanalyzed in parallel for signal comparison. As negative control, aperiplasmic extract of ABD001 was used.

Biacore screening of Z variants: The binding of Z variants to humanIL-1R-I was analyzed in a kinetic screening, using a Biacore 2000instrument, essentially as described in Example 4, but with theexception that Z-ABD (ABD001, SEQ ID NO:1660) periplasmic extracts wereinjected during 2 min. Periplasmic extracts of Z12967, Z16065 and Z16218were included in the screening analysis in duplicates for comparison. Aperiplasmic extract with ABD (ABD001, SEQ ID NO:1660) was included asnegative control.

Results

Sequencing: Sequencing was performed for clones obtained after fourcycles of selection. Each variant was given a unique identificationnumber #####, and individual variants are referred to as Z#####. Theamino acid sequences of the 58 amino acid residues long Z variants arelisted in FIG. 1 and in the sequence listing as SEQ ID NO: 1210-1632.The deduced IL-1R-I binding motifs extend from residue 8 to residue 36in each sequence. The amino acid sequences of the 49 amino acid residueslong polypeptides predicted to constitute the complete three-helixbundle within each of these Z variants extend from residue 7 to residue55.

ELISA screening of Z variants: Clones obtained after four cycles ofselection were produced individually in 96-well plates and were screenedfor human IL-1R-I binding activity in ELISA. 99.5% of the assayed Zvariants gave a positive signal of 2× the blank control or higheragainst 0.2 nM hIL-1R-I-Fc.

EC₅₀ analysis of Z variants: A subset of 102 Z variants displayingresults over 0.74 AU (8.2× the blank control) was subjected to ELISAtarget titration using hIL-1R-I-Fc. Obtained results were used forcalculation of EC₅₀ values (Table 17). The results for Z12967, Z16065and Z16218 were 2.6×10⁻¹⁰, 2.1×10⁻¹⁰ and 2.2×10⁻¹⁰ M, respectively.

TABLE 17 Calculated EC₅₀ values from ELISA titration analysis. SEQ ID Zvariant NO EC₅₀ (M) Z15840 49 2.3 × 10⁻¹⁰ Z15862 71 2.5 × 10⁻¹⁰ Z158761206 1.9 × 10⁻¹⁰ Z15934 1208 1.7 × 10⁻¹⁰ Z15945 150 2.3 × 10⁻¹⁰ Z159751209 1.9 × 10⁻¹⁰ Z16062 266 2.1 × 10⁻¹⁰ Z16183 379 2.3 × 10⁻¹⁰ Z16208404 2.2 × 10⁻¹⁰ Z16603 782 2.2 × 10⁻¹⁰ Z16606 785 2.3 × 10⁻¹⁰ Z187541252 3.5 × 10⁻¹⁰ Z18757 1321 3.0 × 10⁻¹⁰ Z18758 1281 2.4 × 10⁻¹⁰ Z187591314 2.4 × 10⁻¹⁰ Z18760 1435 1.7 × 10⁻¹⁰ Z18763 1431 2.6 × 10⁻¹⁰ Z187661274 3.3 × 10⁻¹⁰ Z18769 1339 2.9 × 10⁻¹⁰ Z18770 1284 2.8 × 10⁻¹⁰ Z187711341 2.5 × 10⁻¹⁰ Z18774 1389 2.6 × 10⁻¹⁰ Z18776 1418 2.2 × 10⁻¹⁰ Z187771326 2.6 × 10⁻¹⁰ Z18785 1323 2.5 × 10⁻¹⁰ Z18786 1320 2.4 × 10⁻¹⁰ Z187901439 2.6 × 10⁻¹⁰ Z18799 1423 2.3 × 10⁻¹⁰ Z18803 1327 2.5 × 10⁻¹⁰ Z188041329 2.7 × 10⁻¹⁰ Z18805 1437 2.7 × 10⁻¹⁰ Z18806 1271 2.7 × 10⁻¹⁰ Z188081342 4.0 × 10⁻¹⁰ Z18810 1278 2.9 × 10⁻¹⁰ Z18811 1405 2.8 × 10⁻¹⁰ Z188131375 2.0 × 10⁻¹⁰ Z18814 1368 2.7 × 10⁻¹⁰ Z18819 1276 2.9 × 10⁻¹⁰ Z188201417 3.3 × 10⁻¹⁰ Z18824 1264 2.8 × 10⁻¹⁰ Z18825 1275 3.5 × 10⁻¹⁰ Z188261356 2.4 × 10⁻¹⁰ Z18827 1318 2.4 × 10⁻¹⁰ Z18831 1308 2.9 × 10⁻¹⁰ Z188321340 1.5 × 10⁻¹⁰ Z18834 1361 2.5 × 10⁻¹⁰ Z18836 1430 2.3 × 10⁻¹⁰ Z188381337 2.4 × 10⁻¹⁰ Z18843 1309 2.8 × 10⁻¹⁰ Z18846 1331 1.9 × 10⁻¹⁰ Z188661324 3.1 × 10⁻¹⁰ Z18874 1421 2.2 × 10⁻¹⁰ Z18875 1420 2.9 × 10⁻¹⁰ Z188771301 3.5 × 10⁻¹⁰ Z18878 1302 3.7 × 10⁻¹⁰ Z18879 1390 3.4 × 10⁻¹⁰ Z188811355 2.8 × 10⁻¹⁰ Z18882 1311 3.1 × 10⁻¹⁰ Z18884 1403 2.3 × 10⁻¹⁰ Z188861432 2.9 × 10⁻¹⁰ Z18887 1386 2.1 × 10⁻¹⁰ Z18888 1422 2.3 × 10⁻¹⁰ Z188891344 2.4 × 10⁻¹⁰ Z18890 1352 2.5 × 10⁻¹⁰ Z18904 1587 3.8 × 10⁻¹⁰ Z189081591 2.4 × 10⁻¹⁰ Z18912 1251 1.4 × 10⁻¹⁰ Z18917 1509 2.2 × 10⁻¹⁰ Z189201270 2.4 × 10⁻¹⁰ Z18966 1272 2.5 × 10⁻¹⁰ Z18967 1388 3.1 × 10⁻¹⁰ Z189681472 2.0 × 10⁻¹⁰ Z18971 1559 3.5 × 10⁻¹⁰ Z18982 1553 2.5 × 10⁻¹⁰ Z190121588 2.4 × 10⁻¹⁰ Z19053 1526 2.4 × 10⁻¹⁰ Z19054 1528 2.5 × 10⁻¹⁰ Z190561586 2.2 × 10⁻¹⁰ Z19057 1558 2.2 × 10⁻¹⁰ Z19059 1240 3.5 × 10⁻¹⁰ Z190601228 2.2 × 10⁻¹⁰ Z19072 1554 1.6 × 10⁻¹⁰ Z19075 1618 1.5 × 10⁻¹⁰ Z190781248 2.2 × 10⁻¹⁰ Z19085 1564 2.1 × 10⁻¹⁰ Z19090 1540 2.0 × 10⁻¹⁰ Z191151489 2.3 × 10⁻¹⁰ Z19117 1631 2.1 × 10⁻¹⁰ Z19118 1579 2.3 × 10⁻¹⁰ Z191231241 2.6 × 10⁻¹⁰ Z19151 1594 2.3 × 10⁻¹⁰ Z19158 1571 1.5 × 10⁻¹⁰ Z191591364 2.1 × 10⁻¹⁰ Z19171 1593 2.3 × 10⁻¹⁰ Z19172 1551 1.6 × 10⁻¹⁰ Z191801393 2.0 × 10⁻¹⁰ Z19181 1549 1.8 × 10⁻¹⁰ Z19182 1628 1.7 × 10⁻¹⁰ Z191831235 2.2 × 10⁻¹⁰ Z19302 1419 2.6 × 10⁻¹⁰ Z19304 1273 2.7 × 10⁻¹⁰ Z193051261 2.6 × 10⁻¹⁰

Biacore screening of Z variants: A subset of 188 IL-1R-I binding Zvariants was submitted to a Biacore kinetic screening. A singleconcentration of hIL-1R-I was injected over each Z-ABD captured fromperiplasmic extracts on a sensor chip surface containing an anti-ABDantibody. The calculated screening affinities are presented in Table 18.The K_(D) values of Z12967, Z16065 and Z16218 were 2.7×10⁻⁸, 7.5×10⁻⁹and 1.3×10⁻⁸ M (average of duplicates), respectively.

Z18814, which has an amino acid sequence that only differs from Z16065in position X₅ of the binding motif BM, has three times higher affinitythan Z-6065. Z16065 has the highest binding affinity (K_(D)) among theIL-1R-I binding Z variants (Table 11) with an alanine residue inposition X₅. Z18814 has an isoleucine residue in position X₅. Several Zvariants with valine or isoleucine in position X₅ were found to havehigh affinity to IL-1R-1.

TABLE 18 Calculated KD values from Biacore kinetic screenina. BiacoreSEQ screening Z variant ID NO K_(D) (M) Z15840 49 6.9 × 10⁻⁹ Z15862 711.3 × 10⁻⁸ Z15876 1206 5.7 × 10⁻⁹ Z15932 1207 5.5 × 10⁻⁹ Z15934 1208 9.4× 10⁻⁹ Z15945 150 9.6 × 10⁻⁹ Z15975 1209 8.9 × 10⁻⁹ Z16062 266 6.4 ×10⁻⁹ Z16183 379 6.5 × 10⁻⁹ Z16191 387 6.3 × 10⁻⁹ Z16208 404 1.1 × 10⁻⁸Z16397 582 1.3 × 10⁻⁸ Z16440 624 1.3 × 10⁻⁸ Z16603 782 9.1 × 10⁻⁹ Z16606785 9.6 × 10⁻⁹ Z16737 910 1.2 × 10⁻⁸ Z18754 1252 2.5 × 10⁻⁹ Z18756 13662.9 × 10⁻⁹ Z18757 1321 3.0 × 10⁻⁹ Z18758 1281 4.8 × 10⁻⁹ Z18759 1314 3.4× 10⁻⁹ Z18760 1435 3.0 × 10⁻⁹ Z18762 1415 2.1 × 10⁻⁹ Z18763 1431 5.2 ×10⁻⁹ Z18766 1274 3.1 × 10⁻⁹ Z18767 12353 5.1 × 10⁻⁹ Z18769 1339 2.7 ×10⁻⁹ Z18770 1284 2.6 × 10⁻⁹ Z18771 1341 3.0 × 10⁻⁹ Z18774 1389 3.0 ×10⁻⁹ Z18776 1418 2.8 × 10⁻⁹ Z18777 1326 3.8 × 10⁻⁹ Z18782 1333 2.7 ×10⁻⁹ Z18783 1257 3.4 × 10⁻⁹ Z18784 1433 3.7 × 10⁻⁹ Z18785 1323 2.7 ×10⁻⁹ Z18786 1320 4.8 × 10⁻⁹ Z18790 1439 3.0 × 10⁻⁹ Z18794 1424 3.7 ×10⁻⁹ Z18796 1352 3.6 × 10⁻⁹ Z18798 1268 3.2 × 10⁻⁹ Z18799 1423 2.6 ×10⁻⁹ Z18800 1328 1.9 × 10⁻⁹ Z18803 1327 3.1 × 10⁻⁹ Z18804 1329 3.3 ×10⁻⁹ Z18805 1437 3.7 × 10⁻⁹ Z18806 1271 4.1 × 10⁻⁹ Z18807 1400 3.8 ×10⁻⁹ Z18808 1342 4.2 × 10⁻⁹ Z18810 1278 3.1 × 10⁻⁹ Z18811 1405 4.5 ×10⁻⁹ Z18813 1375 2.0 × 10⁻⁹ Z18814 1368 2.4 × 10⁻⁹ Z18817 1343 3.5 ×10⁻⁹ Z18819 1276 3.0 × 10⁻⁹ Z18820 1417 3.3 × 10⁻⁹ Z18821 1269 3.5 ×10⁻⁹ Z18823 1282 2.4 × 10⁻⁹ Z18824 1264 3.3 × 10⁻⁹ Z18825 1275 4.9 ×10⁻⁹ Z18826 1356 3.5 × 10⁻⁹ Z18827 1318 4.3 × 10⁻⁹ Z18828 1296 2.6 ×10⁻⁹ Z18830 1295 2.9 × 10⁻⁹ Z18831 1308 3.0 × 10⁻⁹ Z18832 1340 1.6 ×10⁻⁹ Z18833 1297 3.8 × 10⁻⁹ Z18834 1361 2.4 × 10⁻⁹ Z18836 1430 2.9 ×10⁻⁹ Z18838 1337 3.3 × 10⁻⁹ Z18839 1440 3.1 × 10⁻⁹ Z18841 1359 2.5 ×10⁻⁹ Z18843 1309 3.1 × 10⁻⁹ Z18844 1349 4.0 × 10⁻⁹ Z18846 1331 2.1 ×10⁻⁹ Z18847 1363 3.0 × 10⁻⁹ Z18848 1304 3.0 × 10⁻⁹ Z18849 1299 3.0 ×10⁻⁹ Z18850 1367 2.6 × 10⁻⁹ Z18851 1291 3.6 × 10⁻⁹ Z18853 1298 2.3 ×10⁻⁹ Z18856 1330 2.9 × 10⁻⁹ Z18860 1265 6.2 × 10⁻⁹ Z18861 1369 3.3 ×10⁻⁹ Z18863 1362 2.4 × 10⁻⁹ Z18866 1324 2.7 × 10⁻⁹ Z18868 1391 2.7 ×10⁻⁹ Z18870 1428 3.0 × 10⁻⁹ Z18871 1332 3.5 × 10⁻⁹ Z18872 1410 2.6 ×10⁻⁹ Z18873 1306 3.1 × 10⁻⁹ Z18874 1421 2.3 × 10⁻⁹ Z18875 1420 2.7 ×10⁻⁹ Z18876 1286 3.7 × 10⁻⁹ Z18877 1301 2.7 × 10⁻⁹ Z18878 1302 3.8 ×10⁻⁹ Z18879 1390 6.2 × 10⁻⁹ Z18881 1355 3.2 × 10⁻⁹ Z18882 1311 2.6 ×10⁻⁹ Z18884 1403 2.7 × 10⁻⁹ Z18885 1294 3.9 × 10⁻⁹ Z18886 1432 3.9 ×10⁻⁹ Z18887 1386 2.9 × 10⁻⁹ Z18888 1422 2.6 × 10⁻⁹ Z18889 1344 3.4 ×10⁻⁹ Z18890 1352 3.1 × 10⁻⁹ Z18893 1353 2.1 × 10⁻⁹ Z18894 1312 2.5 ×10⁻⁹ Z18895 1411 2.6 × 10⁻⁹ Z18896 1436 3.5 × 10⁻⁹ Z18898 1285 2.5 ×10⁻⁹ Z18904 1587 3.4 × 10⁻⁹ Z18908 1591 3.6 × 10⁻⁹ Z18909 1303 2.7 ×10⁻⁹ Z18910 1416 3.1 × 10⁻⁹ Z18911 1441 2.8 × 10⁻⁹ Z18912 1251 3.6 ×10⁻⁹ Z18916 1536 4.1 × 10⁻⁹ Z18917 1509 7.1 × 10⁻⁹ Z18919 1350 2.8 ×10⁻⁹ Z18920 1270 3.2 × 10⁻⁹ Z18922 1533 4.9 × 10⁻⁹ Z18923 1482 9.0 ×10⁻⁹ Z18925 1479 1.1 × 10⁻⁸ Z18927 1372 3.4 × 10⁻⁹ Z18938 1370 4.5 ×10⁻⁹ Z18941 1409 3.3 × 10⁻⁹ Z18942 1483 1.5 × 10⁻⁸ Z18948 1414 4.3 ×10⁻⁹ Z18952 1290 3.0 × 10⁻⁹ Z18956 1625 1.4 × 10⁻⁸ Z18958 1283 2.8 ×10⁻⁹ Z18959 1357 2.9 × 10⁻⁹ Z18966 1272 2.9 × 10⁻⁹ Z18967 1388 3.2 ×10⁻⁹ Z18968 1472 2.4 × 10⁻⁹ Z18971 1559 5.5 × 10⁻⁹ Z18982 1553 5.2 ×10⁻⁹ Z18989 1592 4.5 × 10⁻⁹ Z19012 1588 4.5 × 10⁻⁹ Z19031 1600 3.1 ×10⁻⁹ Z19043 1505 3.4 × 10⁻⁹ Z19053 1526 8.7 × 10⁻⁹ Z19054 1528 7.0 ×10⁻⁹ Z19056 1586 6.9 × 10⁻⁹ Z19057 1558 4.2 × 10⁻⁹ Z19059 1240 9.2 ×10⁻⁹ Z19060 1228 1.0 × 10⁻⁸ Z19072 1554 5.2 × 10⁻⁹ Z19075 1618 4.8 ×10⁻⁹ Z19078 1248 9.5 × 10⁻⁹ Z19079 1236 1.4 × 10⁻⁸ Z19085 1564 4.9 ×10⁻⁹ Z19090 1540 3.0 × 10⁻⁹ Z19094 1231 1.8 × 10⁻⁸ Z19099 1548 3.8 ×10⁻⁹ Z19107 1566 6.7 × 10⁻⁹ Z19115 1489 6.1 × 10⁻⁹ Z19117 1631 4.2 ×10⁻⁹ Z19118 1579 8.4 × 10⁻⁹ Z19123 1241 1.1 × 10⁻⁸ Z19127 1213 9.8 ×10⁻⁹ Z19135 1247 1.6 × 10⁻⁸ Z19141 1506 1.7 × 10⁻⁸ Z19143 1471 1.4 ×10⁻⁹ Z19144 1374 3.1 × 10⁻⁹ Z19146 1434 2.9 × 10⁻⁹ Z19147 1307 2.4 ×10⁻⁹ Z19151 1594 2.7 × 10⁻⁹ Z19153 1310 3.1 × 10⁻⁹ Z19158 1571 1.5 ×10⁻⁹ Z19159 1364 1.9 × 10⁻⁹ Z19160 1379 3.8 × 10⁻⁹ Z19166 1305 3.5 ×10⁻⁹ Z19168 1500 4.6 × 10⁻⁹ Z19169 1402 2.7 × 10⁻⁹ Z19170 1334 2.0 ×10⁻⁹ Z19171 1593 3.0 × 10⁻⁹ Z19172 1551 4.5 × 10⁻⁹ Z19180 1393  7.9 ×10⁻¹⁰ Z19181 1549 9.7 × 10⁻⁹ Z19182 1628 2.8 × 10⁻⁹ Z19183 1235 1.2 ×10⁻⁸ Z19302 1419 3.2 × 10⁻⁹ Z19303 1262 4.0 × 10⁻⁹ Z19304 1273 4.5 ×10⁻⁹ Z19305 1261 3.8 × 10⁻⁹

Example 9 Production and Characterization of Z Variants from the Firstand Second Matured Libraries

This Example describes the general procedure for subcloning andproduction of His₆-tagged Z variants originating from the first andsecond maturation library phage selections, characterization of theirtarget binding, target blocking and melting points.

Materials and Methods

Subcloning of Z variants with a His₆ tag: A subset of Z variantsscreened in Example 8 was chosen for subcloning. The DNA of therespective Z variant was amplified from the phage library vectorpAY02592 and was subcloned with an N-terminal His₆ tag using standardmolecular biology techniques essentially as described in Example 2.

Cultivation: E. coli T7E2 cells (GeneBridges) were transformed withplasmids containing the gene fragment of each respective IL-1R-I bindingZ variant. The resulting recombinant strains were cultivated in mediasupplemented with 50 μg/ml kanamycin at 30° C. in 50 ml scale using theEnPresso protocol (BioSilta). In order to induce protein expression,IPTG was added to a final concentration of 0.2 mM at OD₆₀₀≈10. Afterinduction, the cultivations were incubated for 16 h. The cells wereharvested by centrifugation.

Purification of IL-1R-I binding Z variants with a His₆ tag:Approximately 2/3 of the IL-1R-I binding Z variants were purified asdescribed in Example 2 but without the buffer exchange between the IMACand RPC purification steps. The linear gradient during RPC purificationwas also changed to 0-60% RPC solvent B for 18 ml. The remaining 1/3 ofthe IL-1R-I binding Z variants were only IMAC purified before the finalbuffer exchange to PBS. Purification was essentially performed asdescribed in the first part of Example 2. Approximately 1-2 g of eachcell pellet was used.

Biacore kinetic analysis: The kinetic constants (k_(on) and k_(off)) andaffinities (K_(D)) for human and cynomolgus IL-1R-I were determined forHis₆-tagged Z variants using a Biacore 2000 instrument (GE Healthcare).The experiment was run essentially as described in Example 5, using 48,12, 3 and 0.75 nM of the Z variants. The analyte injection time was 3min followed by 6 min dissociation and 2×5 s glycine pH 3.0,supplemented with 0.5 M NaCl, was used for regeneration of the surfacesbetween the cycles. Kinetic constants were calculated from the obtainedsensorgrams of three or four concentrations of the respective Z variant,using a 1:1 binding model in the BiaEvaluation software 4.1 (GEHealthcare). His₆-tagged Z12967 (SEQ ID NO:9), Z16065 (SEQ ID NO:268),Z16163 (SEQ ID NO:359) and Z18557 (SEQ ID NO:1205) were included in theassay for comparison.

In vitro IL-1β neutralization assay: His₆-tagged IL-1R-I specific Zvariants were tested for their inhibitory capacity in the TF-1 cellassay. The assay was run as described in Example 2. The data on cellgrowth was assessed by non-linear regression to a four-parameterdose-response curve, and IC₅₀ values were determined using GraphPadPrismprogram. His₆-tagged Z12967, Z16065, Z16163 and Z18557 were included forcomparison.

Circular dichroism (CD) spectroscopy analysis: CD was analyzed forHis₆-tagged Z variants, as described in Example 2.

Results

Production of His₆-tagged Z variants: The IL-1R-I binding Z variantswith a His₆ tag were expressed as soluble gene products in E. coli. Theamount of purified protein was determined spectrophotometrically bymeasuring the absorbance at 280 nm and ranged from approximately 0.4 to7 mg protein per g pellet for IL-1R-I binding Z variants which were bothIMAC and RPC purified. The amount of purified protein ranged fromapproximately 4 to 15 mg protein per g pellet for IL-1R-I binding Zvariants which were only IMAC purified. SDS-PAGE analysis of each finalprotein preparation showed that these predominantly contained theIL-1R-I binding Z variant. The correct identity and molecular weight ofeach Z variant were confirmed by HPLC-MS analysis.

Biacore kinetic analysis: The interactions of His₆-taggedIL-1R-1-binding Z variants with human and cynomolgus IL-1R-I wereanalyzed in a Biacore instrument by injecting various concentrations ofthe Z variants over a surface containing immobilized IL-1R-1. A summaryof the calculated kinetic parameters (K_(D), k_(on) and k_(off)) for thehuman IL-1R-I binding is given in Table 19. The K_(D) values of Z12967,Z16065, Z16163 and Z18557 were 8.2×10⁻¹⁰, 5.1×10⁻¹⁰, 7.6×10⁻¹⁰ and1.4×10⁻¹⁰ M, respectively. The K_(D) values of cynomolgus IL-1R-1binding were 2.0×10⁻⁹, 6.7×10⁻⁹ and 3.3×10⁻⁹ M for Z18754, Z18760 andZ18800, respectively.

TABLE 19 Kinetic parameters and affinities for His₆-Z polypeptidesbinding to human IL-1R-I. Z variant SEQ ID NO k_(on) (1/Ms) k_(off)(1/s) K_(D) (M) Z15876 1206 5.2 × 10⁶ 4.1 × 10⁻³ 8.0 × 10⁻¹⁰ Z15932 12071.8 × 10⁷ 1.1 × 10⁻² 6.4 × 10⁻¹⁰ Z15934 1208 1.4 × 10⁷ 1.1 × 10⁻² 7.5 ×10⁻¹⁰ Z15975 1209 4.1 × 10⁷ 2.9 × 10⁻² 7.1 × 10⁻¹⁰ Z18754 1252 7.0 × 10⁷6.0 × 10⁻³ 8.6 × 10⁻¹¹ Z18760 1435 1.3 × 10⁷ 2.7 × 10⁻³ 2.1 × 10⁻¹⁰Z18762 1415 4.2 × 10⁷ 5.2 × 10⁻³ 1.3 × 10⁻¹⁰ Z18769 1339 1.9 × 10⁷ 3.1 ×10⁻³ 1.6 × 10⁻¹⁰ Z18770 1284 3.0 × 10⁷ 5.0 × 10⁻³ 1.7 × 10⁻¹⁰ Z187991423 2.6 × 10⁷ 4.5 × 10⁻³ 1.8 × 10⁻¹⁰ Z18800 1328 1.8 × 10⁷ 2.5 × 10⁻³1.4 × 10⁻¹⁰ Z18813 1375 2.0 × 10⁷ 3.9 × 10⁻³ 2.0 × 10⁻¹⁰ Z18814 1368 3.0× 10⁷ 4.4 × 10⁻³ 1.5 × 10⁻¹⁰ Z18817 1343 2.8 × 10⁷ 4.8 × 10⁻³ 1.7 ×10⁻¹⁰ Z18823 1283 4.7 × 10⁶ 1.8 × 10⁻³ 3.8 × 10⁻¹⁰ Z18831 1308 4.0 × 10⁷5.3 × 10⁻³ 1.3 × 10⁻¹⁰ Z18832 1340 5.9 × 10⁷ 7.0 × 10⁻³ 1.2 × 10⁻¹⁰Z18834 1361 5.8 × 10⁷ 8.0 × 10⁻³ 1.4 × 10⁻¹⁰ Z18846 1331 4.7 × 10⁷ 5.1 ×10⁻³ 1.1 × 10⁻¹⁰ Z18850 1367 1.3 × 10⁷ 2.3 × 10⁻³ 1.7 × 10⁻¹⁰ Z188531298 2.2 × 10⁷ 3.0 × 10⁻³ 1.4 × 10⁻¹⁰ Z18856 1330 4.6 × 10⁷ 5.7 × 10⁻³1.2 × 10⁻¹⁰ Z18863 1362 2.2 × 10⁷ 4.8 × 10⁻³ 2.2 × 10⁻¹⁰ Z18866 1324 3.6× 10⁷ 4.2 × 10⁻³ 1.2 × 10⁻¹⁰ Z18874 1421 4.9 × 10⁷ 5.2 × 10⁻³ 1.1 ×10⁻¹⁰ Z18875 1420 2.4 × 10⁷ 2.8 × 10⁻³ 1.2 × 10⁻¹⁰ Z18882 1311 1.2 × 10⁷3.0 × 10⁻³ 2.5 × 10⁻¹⁰ Z18888 1422 1.8 × 10⁷ 2.9 × 10⁻³ 1.6 × 10⁻¹⁰Z18893 1353 8.6 × 10⁶ 2.4 × 10⁻³ 2.7 × 10⁻¹⁰ Z18898 1285 6.7 × 10⁷ 4.4 ×10⁻³ 6.6 × 10⁻¹¹ Z18912 1251 9.2 × 10⁷ 1.9 × 10⁻² 2.1 × 10⁻¹⁰ Z189201270 3.6 × 10⁷ 5.5 × 10⁻³ 1.5 × 10⁻¹⁰ Z18968 1472 3.0 × 10⁷ 8.4 × 10⁻³2.8 × 10⁻¹⁰ Z19072 1554 4.3 × 10⁷ 1.8 × 10⁻² 4.1 × 10⁻¹⁰ Z19075 1618 1.4× 10⁷ 6.4 × 10⁻³ 4.5 × 10⁻¹⁰ Z19143 1471 1.7 × 10⁷ 3.9 × 10⁻³ 2.2 ×10⁻¹⁰ Z19147 1307 2.8 × 10⁷ 4.3 × 10⁻³ 1.5 × 10⁻¹⁰ Z19151 1594 2.8 × 10⁷7.0 × 10⁻³ 2.5 × 10⁻¹⁰ Z19158 1571 2.7 × 10⁷ 4.4 × 10⁻³ 1.6 × 10⁻¹⁰Z19159 1364 2.7 × 10⁷ 5.0 × 10⁻³ 1.9 × 10⁻¹⁰ Z19170 1334 2.1 × 10⁷ 4.3 ×10⁻³ 2.0 × 10⁻¹⁰ Z19172 1551 4.9 × 10⁷ 2.3 × 10⁻² 4.6 × 10⁻¹⁰ Z191801393 2.2 × 10⁷ 3.1 × 10⁻³ 1.4 × 10⁻¹⁰ Z19181 1549 2.0 × 10⁷ 1.6 × 10⁻²8.2 × 10⁻¹⁰ Z19182 1628 2.1 × 10⁷ 5.5 × 10⁻³ 2.6 × 10⁻¹⁰

In vitro IL-1β neutralization assay: The IL-1β inhibition ability ofHis₆-tagged IL-1R-1-binding Z variants was analyzed in a TF-1 cellassay. The resulting IC₅₀ values are presented in Table 20. The IC₅₀values of Z12967, Z16065, Z16163 and Z18557 were 7.4, 3.3, 3.3 and 0.6nM, respectively.

TABLE 20 IC₅₀ values for His₆-Z polypeptides from a TF-1 cell assay. Zvariant SEQ ID NO IC₅₀ (nM) Z15934 1208 6.4 Z15975 1209 3.3 Z18754 12520.6 Z18760 1435 0.9 Z18813 1375 1.2 Z18814 1368 0.4 Z18831 1308 0.4Z18832 1340 0.5 Z18834 1361 0.5 Z18846 1331 0.2 Z18874 1421 0.3 Z189121251 1 Z18968 1472 1.4 Z19072 1554 2.4 Z19159 1364 1.2 Z19172 1551 2Z19181 1549 4.6 Z19182 1628 1.5

CD analysis: The CD spectra determined for the IL-1R-I binding Zvariants with a His₆ tag showed that each had an a-helical structure at20° C. This result was also verified in the variable temperaturemeasurements, wherein melting temperatures were determined (Table 21).Reversible folding was seen for all the IL-1R-I binding Z variants whenoverlaying spectra measured before and after heating to 90° C.

The thermo-stability and the a-helical content of the IL-1R-I binding Zvariants having a valine or isoleucine residue in position X₅ of the BM(Z18912, Z18920, Z18968, Z19072, Z19075, Z19143, Z19147, Z19151, Z19158,Z19159, Z19170, Z19172, Z19180, Z19181 and Z19182) were notsubstantially different from the Z variants having an alanine residue inposition X₅.

TABLE 21 Melting temperatures (Tm). Z variant SEQ ID NO Tm (° C.) Z158761206 55 Z15932 1207 60 Z15934 1208 62 Z15975 1209 59 Z18754 1252 63Z18760 435 57 Z18762 1415 62 Z18769 1339 59 Z18770 1284 59 Z18799 142360 Z18800 1328 67 Z18813 1375 61 Z18814 1368 61 Z18817 1343 60 Z188231283 52 Z18831 1308 58 Z18832 1340 58 Z18834 1361 58 Z18846 1331 60Z18850 1367 62 Z18853 1298 59 Z18856 1330 61 Z18863 1362 57 Z18866 132461 Z18874 1421 61 Z18875 1420 61 Z18882 1311 53 Z18888 1422 61 Z188931353 60 Z18898 1285 59 Z18912 1251 53 Z18920 1270 56 Z18968 1472 62Z19072 1554 63 Z19075 1618 56 Z19143 1471 56 Z19147 1307 57 Z19151 159459 Z19158 1571 55 Z19159 1364 54 Z19170 1334 52 Z19172 1551 59 Z191801393 58 Z19181 1549 61 Z19182 1628 62

Example 10 Production and Characterization of IL-1R-I Binding FusionProteins

This Example describes the common steps for DNA construction andproduction of different IL-1R-I binding proteins variants fused todifferent in vivo half-life extending fusion partners.

Materials and Methods

DNA construction: DNA encoding a set of IL-1R-I binding Z variants (seebelow) fused to human IgG1 Fc (SEQ ID NO:1662), an albumin bindingpolypeptide variant (PP013; SEQ ID NO:1661), human albumin (SEQ IDNO:1663) or human transferrin (SEQ ID NO:1664), optionally via differentlinkers (Table 22), were codon optimized for expression in E. coli orfor expression in Chinese hamster ovary (CHO) cells and synthesized bythe Invitrogen GeneArt Gene Synthesis service at Thermo FisherScientific. The genes were cloned in expression vectors for subsequentexpression in E. coli or in CHO cells.

The fusion proteins were based on the binding motifs of the IL-1R-Ibinding Z variants a) Z12967 (SEQ ID NO:9), b) Z12895 (SEQ ID NO:12), c)Z18831 (SEQ ID NO:1308), d) Z18846 (SEQ ID NO:1331), e) Z18754 (SEQ IDNO:1252), f) Z19151(SEQ ID NO:1594), g) Z18874 (SEQ ID NO:1421), h)Z18760 (SEQ ID NO:1435), i) Z18800 (SEQ ID NO:1328), j) Z18898 (SEQ IDNO:1285) and k) Z19147 (SEQ ID NO:1307) and the different fusionspartners described above. The IL-1R-I binding Z variants a)-k) containeddifferent scaffold mutations, (positions 1, 2, 23, 52 and/or 53) or adeletion combined with mutations (deletion at positions 1-3; AV1 D2A3,mutations in positions 52 and 53), see Table 22 below. The mutated Zvariants are listed in FIG. 1 as SEQ ID NO:1667-1668 and 1670-1679.These obtained fusion polypeptides were PS10405 (SEQ ID NO:1639),PS10407 (SEQ ID NO:1640), PS10411 (SEQ ID NO:1641), PS10412 (SEQ IDNO:1642), PS10415 (SEQ ID NO:1643), PS10416 (SEQ ID NO:1644), PS10417(SEQ ID NO:1645), PS10534 (SEQ ID NO:1646), PS10535 (SEQ ID NO:1647),PS10536 (SEQ ID NO:1648), PS10537 (SEQ ID NO:1649), PS10538 (SEQ IDNO:1650), PS10539 (SEQ ID NO:1651), PS10574 (SEQ ID NO:1652), PS10575(SEQ ID NO:1653), PS10576 (SEQ ID NO:1654), PS10580 (SEQ ID NO:1655),PS10581 (SEQ ID NO:1656), PS10582 (SEQ ID NO:1657), see Table 22.

TABLE 22 Fusion proteins and expression systems Fusion Expression SEQ IDNO proteins Construct system 1639 PSI0405 Z12967[V1A; D2E; N52S;D53E]-VEGS-ABD E. coli 1640 PSI0407 Z12895[V1A; D2E; N52S;D53E]-VEGS-ABD E. coli 1641 PSI0411 AS-ABD-GS-Z12967[V1A; D2E; N52S;D53E] E. coli 1642 PSI0412 AS-ABD-(G₄S)₂- E. coli Z12967[V1A; D2E; N52S;D53E] 1643 PSI0415 AS-ABD-Z12967[V1A; D2E; N52S; D53E] E. coli 1644PSI0416 AS-ABD-Z12967[ΔV1D2A3; N52S; D53E] E. coli 1645 PSI0417AS-ABD-((KEAAA)₃KELAA)₂- E. coli Z12967[V1A; D2E; N52S; D53E] 1646PSI0534 Z18831[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1647PSI0535 Z18846[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1648PSI0536 Z18754[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1649PSI0537 Z19151[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1650PSI0538 Z18874[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1651PSI0539 Z18760[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1652PSI0574 Z18800[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1653PSI0575 Z18898[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1654PSI0576 Z19147[V1A; D2E; N52S; D53E]-AS(G₄S)₂- E. coli IgG1 Fc 1655PSI0580 Z18754[V1A; D2E; N52S; D53E]-AS(G₄S)₂- CHO Albumin 1656 PSI0581Z18754[V1A; D2E; T23N; N52S; D53E]- CHO AS(G₄S)₂-Albumin 1657 PSI0582Z18754[V1A; D2E; N52S; D53E]-AS(G₄S)₂- CHO Transferrin 1658 PSI0589PSI0536[amino acids 1-86], dimeric

Cultivation and purification of fusion proteins: E. coli cells weretransformed with expression vectors containing the gene fragmentscorresponding to the IL-1R-I binding fusion proteins, see Table 22, andthen cultivated in bioreactors using fed-batch techniques or in shakeflasks, followed by protein expression and harvest of cells bycentrifugation. Cell pellets were stored at −20° C. Expression ofIL-1R-I binding Z variants, see Table 22, were also performed using theExpiCHO expression system (Thermo Fisher Scientific), essentiallyaccording to the manufacturer's protocol. Supernatants were harvested bycentrifugation 12 days after transfection of expression vectors andstored at −70° C.

Frozen E. coli cell pellets were resuspended and then disrupted bysonication and the cell debris subsequently removed by centrifugationfollowed by filtration (0.22 μm). The frozen supernatants from theExpiCHO cultures were thawed and filtrated (0.22 μm). Each supernatant,containing the IL-1R-I binding Z variants was purified usingconventional chromatography methods, such as affinity chromatography,ion exchange chromatography, hydrophobic interaction chromatography andsize exclusion chromatography. Z variants for use in animal studies werealso subjected to an endotoxin removal purification using Detoxi-GelEndotoxin Removing Columns (Pierce, cat.no. 20344). Purified Z variantswere buffer exchanged to PBS and, unless otherwise stated, PBS was alsothe formulation buffer used in subsequent experiments. The purity of theIL-1R-I binding Z variants was analyzed by SDS-PAGE stained withCoomassie Blue and the molecular weight of each purified Z variant wasanalyzed using mass spectrometry (HPLC/MS or MALDI-TOF/MS).

The IL-1R-I binding fusion protein PS10589 (SEQ ID NO:1658), a dimericZ18754 variant linked in the hinge region by disulfides, was obtained byremoving the IgG1 Fc part from PS10536, see Table 22. This was achievedby incubation of purified PS10536 with the IdeS protease (FabRICATOR,Genovis AB). The cleavage products were then purified using conventionalchromatography methods such as ion exchange chromatography and affinitychromatography. The purified dimeric Z18754 variant was subjected tobuffer exchange into PBS, which was also the formulation buffer used insubsequent experiments. The purity was analyzed by SDS-PAGE stained withCoomassie Blue and the molecular weight was analyzed using massspectrometry (HPLC/MS or MALDI-TOF/MS).

Biolayer Interferometry (BLI) kinetic analysis: Binding of four IL-1R-Ibinding fusion proteins to human and cynomolgus IL-1R-I was analyzedusing BLI. The Fc fused IL-1R-I binding Z variants denoted PS10536,PS10537, PS10534 and PS10535 (SEQ ID NO:1648, 1649, 1646, 1647) wereloaded onto a Protein A sensor (Pall, ForteBio cat. no. 18-5010) at 0.5μg/ml for 150 s in each cycle using an OctetRED96 instrument (Pall,ForteBio). Subsequently, these sensors were exposed to different andincreasing concentrations of human IL-1R-I (R&D systems cat. no.269-1R/CF) ranging from 0.15 to 5 nM or cynomolgus IL-1R-I (SEQ IDNO:I665) ranging from 2.5 to 80 nM. Association was recorded for 300 sand dissociation for 300 s (human) or 90 s (cynomolgus) and spectrogramswere recorded and analyzed according to a 1:1 Langmuir model usingglobal fit (Octet System Data Analysis 8.2 software, Pall, ForteBio).Each sensor was referenced to a sensor with the same ligand loaded ontoit but exposed to buffer only. Following each association/dissociationcycle, all sensors were regenerated by three pulses of 10 mM glycine atpH 2.5 for 10 s each. All steps were carried out at 25° C. at a shakingspeed of 1000 rpm and all dilutions of fusion proteins (Z-Fc fusionproteins) and analytes (human or cynomolgus IL-1R-1) were in 1×kineticsbuffer (Pall, ForteBio).

Results

Cultivation and purification: All the IL-1R-I binding fusion proteins(Table 22) were expressed to high levels in E. coli or CHO cells assoluble proteins. Purification resulted in protein preparations withhigh purity, which was analyzed by SDS-PAGE stained with Coomassie Blue.The correct identity and molecular weight of each Z variant wereconfirmed by mass spectrometry analysis.

BLI kinetic analysis: Four Z-Fc fusion protein variants (SEQ ID NO:1648, 1649, 1646, 1647) were analyzed for the ability to bind human andcynomolgus IL-1R-I loaded onto protein A sensor using BLI technology onan OctetRED96 instrument. All four variants bound IL-1R-I according to a1:1 model with B-max values ranging from 0.46 to 0.72 nm (human) or 0.31to 0.37 nm (cynomolgus). The kinetic data; association rate constant(k_(on)), dissociation rate constant (k_(off)) and dissociation constantK_(D) for the Z-Fc fusion protein variants are presented in Table 23.

TABLE 23 Kinetic parameters for IL-1R-I binding fusion proteins Fusionproteins and Human IL-1R-I Cynomolgus IL-1R-I SEQ ID NO K_(D) (M) k_(on)(1/Ms) k_(off) (1/s) K_(D) (M) k_(on) (1/Ms) k_(off) (1/s) PSI0536 (SEQ7.5 × 10⁻¹⁰ 8.99 × 10⁵ 6.79 × 10⁻⁴ 1.4 × 10⁻⁸ 1.70 × 10⁶ 2.34 × 10⁻² IDNO: 1648) PSI0537 (SEQ 1.2 × 10⁻⁹  8.41 × 10⁵ 1.02 × 10⁻³ 3.1 × 10⁻⁸1.78 × 10⁶ 5.48 × 10⁻² ID NO: 1649) PSI0534 (SEQ 1.6 × 10⁻⁹  4.04 × 10⁵6.42 × 10⁻⁴ 2.8 × 10⁻⁸ 1.08 × 10⁶ 2.99 × 10⁻² ID NO: 1646) PSI0535 (SEQ7.2 × 10⁻¹⁰ 5.83 × 10⁵ 4.21 × 10⁻⁴ 1.1 × 10⁻⁸ 1.49 × 10⁶ 1.66 × 10⁻² IDNO: 1647)

Example 11 In Vitro Pharmacological Activity Analysis Usinq a Cell-BasedAssay Materials and Methods

The inhibitory effect of IL-1R-I binding fusion proteins on IL-1βinduced IL-6 production in Normal Human Dermal Fibroblasts (NHDF) cellswas monitored.

Cells were seeded three days prior to treatment with proteins. Proteins(IL-1R-I binding fusion proteins or anakinra) were diluted to a startingconcentration of 100 nM and subsequently serially 1:4 nine timesresulting in a concentration range of 100 nM to 0.38 μM in serum-freegrowth medium in the presence of 9 μM recombinant human serum albumin(rHSA). The IL-1R-I binding fusion proteins or anakinra were tested inpresence of a challenge dose of 3.4 pM IL-1β and the cells wereincubated for 22 hours with proteins at 37° C., followed by harvestingof medium. Harvested medium was diluted 41× before IL-6 content wasanalyzed using a human IL-6 ELISA kit (R&D Systems) according tomanufacturer's recommendations. Data was analyzed using XLfit and IC₅₀values were calculated from concentration-response curves.

The inhibitory effect of His₆-tagged IL-1R-I binding Z variants,produced as described in Example 9, on IL-1β induced IL-6 production inNHDF cells was similarly monitored and IC₅₀ values calculated.

Chemically synthesized protein: A chemically synthesized version of SEQID NO:1672, denoted PS10558, was ordered from BACHEM AG. The activity ofPS10558 was assessed according to the method set out above.

Results

The IL-1β induced IL-6 release from NHDF cells was reduced in aconcentration-dependent manner by the IL-1R-I binding fusion proteins aswell as by anakinra. Each IL-1R-I binding fusion protein was testedtwice and the data from both experiments along with historical averageof anakinra (n>20) in this assay are presented in Table 24 and one ofthe two experiments is also presented in FIG. 2.

TABLE 24 In vitro IC₅₀ values Denotation SEQ ID NO IC₅₀ (pM) PSI05361648 66; 45 PSI0537 1649 260; 200 PSI0534 1646 140; 110 PSI0535 1647 80;50 PSI0538 1650 87; 70 PSI0539 1651 240; 110 anakinra Average 70Some IL-1R-I binding fusion proteins were tested in the same manner butonly one time. The results from this experiment are presented in Table25.

TABLE 25 In vitro IC₅₀ values Denotation SEQ ID NO IC₅₀ (pM) PSI05891658 1.8 PSI0582 1657 2.0 PSI0580 1655 1500 PSI0581 1656 1800

The IL-1β inhibition ability of His₆-tagged IL-1R-I-binding Z variants,previously demonstrated in a TF-1 cell assay (Example 9, Table 20), wasconfirmed in this NHDF assay (results not shown).

The IC₅₀ value of the chemically synthesized PS10558 was 690 μM.

Example 12 In Vitro Pharmacological Activity Analysis Using a WholeBlood Assay

The inhibitory effect of IL-1R-I binding fusion proteins on IL-1βinduced IL-6 production in whole blood from individual donors wasmeasured.

Materials and Methods

Blood from healthy donors was collected 1-3 hours prior to treatmentwith the IL-1R-I binding fusion proteins (SEQ ID NO:1648-1649). Proteins(IL-1R-I binding fusion proteins or anakinra) were diluted to startingconcentration of 500 nM in serum free RPMI (ThermoFisher Scientific,RPMI 1640 Medium, GlutaMAX™ Supplement, Cat. No.: 61870010) andsubsequently serially diluted 1:3 in a ten-step dilution seriesresulting in a concentration range of 500 nM to 25 pM. The IL-1R-Ibinding fusion polypetides or anakinra were tested in presence of achallenge dose of 100 pM IL-1β. The blood was incubated for 21-23 hourswith proteins, followed by harvesting of plasma. Harvested plasma wasdiluted 2× and IL-6 content was analyzed by using a human IL-6 kit(V-PLEX Human IL-6 Kit Human interleukin-6, MSD) according tomanufacturer's recommendations.

IL-6 concentrations (pg/ml) were calculated using a standard curve(provided by the kit) and data was analyzed using XLfit and IC₅₀ valueswere calculated from the concentration-response curves.

Results

The IL-1β induced IL-6 release in human whole blood was reduced in aconcentration-dependent manner by the IL-1R-I binding fusion proteins aswell as by anakinra. IL-1R-I binding fusion proteins were tested inblood from five to six individual healthy donors and compared toanakinra data from ten individual blood donors (FIG. 3). The median IC₅₀values were 0.32, 1.25 and 0.31 nM for SEQ ID NO:1648 (PS10536), SEQ IDNO:1649 (PS10537) and anakinra, respectively.

Example 13 In Vivo Pharmacokinetics of IL-IR-1 Binding Fusion Proteinsin Rats

In this Example, the pharmacokinetics of IL-1R-1-binding fusion proteins(SEQ ID NO:1646-1651) were evaluated in a single dose study in malerats.

Material and Methods

Fusion proteins: The IL-1R-I binding fusion proteins PS10534, PS10535,PS10536, PS10537, PS10538 and PS10539 (see Table 22, SEQ IDNO:1646-1651) were evaluated in this study. All six test items wereconstituted as a solution in 25 mM sodium phosphate and 125 mM sodiumchloride, pH 7.0.

In-life phase: The pharmacokinetic properties of the six fusion proteinswere investigated in male Sprague-Dawley rats. For each test item, threerats were given an i.v. single dose of 15 mg/kg (2.5 ml/kg) injected inthe lateral tail vein and another three rats were given a s.c. singledose of 30 mg/kg (5 ml/kg) injected in the neck region. Blood samplesfor serum preparation were collected pre dosing and at 5 and 20 min, 1,4, 8, 24, 48, 72 and 96 hrs post i.v. dosing, or 20 min, 1, 4, 8, 24,48, 72, 96 and 120 hrs post s.c. dosing.

Quantitative ELISA: Determination of fusion protein levels in rat serumsamples was performed by enzyme-linked immunosorbent assay (ELISA). TheELISA assay employed the quantitative sandwich enzyme immunoassaytechnique.

In brief; a goat polyclonal anti-Z antibody recognizing the Z variantdomain of the IL-1R-I binding fusion protein (produced in house,Affibody AB) was coated onto a microplate. Unbound polyclonal antibodywas washed away and casein was added as blocking agent to reduceunspecific binding to the plastic surface. After removal of unboundcasein by a second wash step, standards and samples were pipetted to thewells and any fusion protein present was bound to the immobilizedantibody. After washing away any unbound substances, a HRP labled Swineanti-rabbit antibody (Dako, cat. no. P0399) was added. Following a washto remove any unbound anti-rabbit HRP reagent, a substrate solution wasadded to the wells and color developed in proportion to the amount offusion protein bound in the initial step. The color development wasstopped and the intensity of the color was measured.

Pharmacokinetic analysis: The pharmacokinetic analysis was based onmedian serum concentration versus time data from each dose group. Theobserved maximum concentration (C_(max)) and the time to maximum serumconcentration (t_(max)) were taken directly from the bioanalytical data.Other pharmacokinetic parameters, i.e. clearance (CL), apparentclearance following s.c. administration (CL/F), apparent volume ofdistribution at steady-state (V_(ss)), mean residence time (MRT) andterminal half-life (t_(1/2z)), were estimated by non-compartmentalanalysis using Phoenix WinNonlin software version 6.3 (Pharsight Corp.,USA). Estimation of the terminal slope following i.v. dosing was basedon four data points, from 24 to 96 hrs. Estimation of the terminal slopefollowing s.c. dosing was based on five data points, from 24 to 120 hrs.Calculation of the subcutaneous bioavailability (F) was performed usingMicrosoft Excel.

Results

There was no significant difference in the pharmacokinetics between thesix tested fusion proteins. The pharmacokinetics following i.v.administration was characterized by a clearance in the order of 2.4ml/h·kg and a volume of distribution of about 140 ml/kg, translatinginto a mean residence time of ca 60 hrs (Table 26).

TABLE 26 Pharmacokinetic parameter estimates following an intravenoussingle dose administration, 15 mg/kg, to male rats Parameter PSI0534PSI0535 PSI0536 PSI0537 PSI0538 PSI0539 CL (ml/h · kg) 2.83 2.40 2.682.06 2.36 2.03 V_(ss) (ml/kg) 159 149 143 136 146 113 MRT (hrs) 56.462.1 53.5 65.9 61.6 55.5 t_(1/2z) (hrs) 48.7 52.1 44.7 55.1 50.4 48.3The subcutaneous bioavailability was on average 50% and peak levels wereobserved at 24 hrs after dosing. The serum levels then declined with ahalf-life ranging from 45 to 59 hrs (Table 27).

TABLE 27 Pharmacokinetic parameter estimates following a subcutaneoussingle dose administration, 30 mg/kg, to male rats Parameter PSI0534PSI0535 PSI0536 PSI0537 PSI0538 PSI0539 F (%) 41.5 49.6 46.7 60.3 47.252.2 C_(max) (nM) 828 866 948 1115 796 886 t_(max) (hrs) 24 24 24 24 2424 CL/F (ml/h · kg) 6.81 5.70 6.05 4.69 5.98 5.42 V_(z)/F (ml/kg) 445457 403 372 509 424 t_(1/2z) (hrs) 45.3 55.6 46.1 55.1 59.0 54.3 MRT(hrs) 72.4 87.3 74.5 87.4 91.7 86.2

Example 14 In Vivo Pharmacokinetics of an IL-1R-I Binding Fusion Proteinin Monkeys Material and Methods

Fusion proteins: The IL-1R-I binding fusion protein denoted PS10536 (SEQID NO:1648) was constituted as a solution in 25 mM sodium phosphate and125 mM sodium chloride, pH 7.0.

In-life phase: The pharmacokinetic properties of PS10536 wereinvestigated in naive male Cynomolgus monkeys. Three monkeys were givenan i.v. single dose of 5 mg/kg (1 ml/kg) injected in the tail vein andanother three monkeys were given a s.c. single dose of 10 mg/kg (1ml/kg) injected in the dorsal region. Blood samples for serumpreparation were collected pre dosing and at 5 and 20 min, 1, 2, 4, 8,12, 24, 48, 72, 96, 120, 144, 168, 240 and 504 hrs post i.v. dosing, or20 min, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 240 and 504 hrspost s.c. dosing.

Quantitative ELISA: Quantification of PS10536 in serum from monkeys wasperformed according to the ELISA described in Example 13.

Pharmacokinetic analysis: Pharmacokinetic parameters were based onindividual serum concentration versus time data and determined usingWinNonlin software as described in Example 13. Estimation of theterminal slope following i.v. dosing was based on seven data points,from 72 to 504 hrs. Estimation of the terminal slope following s.c.dosing was based on eight data points, from 48 to 504 hrs. Calculationof the subcutaneous bioavailability (F) was performed using MicrosoftExcel.

Results

The pharmacokinetics of PS10536 (SEQ ID NO:1648) following i.v.administration was characterized by a low clearance (1.07 ml/h·kg) and asmall volume of distribution (117 ml/kg), translating into a meanresidence time of 109 hrs (Table 28).

TABLE 28 Mean (±SD) pharmacokinetic parameter estimates of PSI0536following an intravenous single dose of 5.04 (±0.01) mg/kg to three malemonkeys Parameter Estimate CL (ml/h · kg)  1.07 ± 0.04 V_(ss) (ml/kg)117 ± 3 MRT (hrs) 109 ± 1 t_(1/2z) (hrs)  92.7 ± 3.7The mean subcutaneous bioavailability of PS10536 was 73.9%. Peak levelswere observed at an average of 18.7 hrs after dosing. The serum levelsthen declined with a half-life of 77.3 hrs (Table 29).

TABLE 29 Mean (+SD) pharmacokinetic parameter estimates of PSI0536following a subcutaneous single dose of 10.1 (+0.1) mg/kg to three malemonkeys Parameter Estimate F (%) 73.9 ± 5.5 C_(max) (nM)  779 ± 159t_(max) (hrs) 18.7 ± 7.5 CL/F (ml/h · kg)  1.46 ± 0.10 V_(z)/F (ml/kg)163 ± 23 t_(1/2z) (hrs) 77.3 ± 6.3 MRT (hrs) 115 ± 14

Conclusion

Assuming a minimum target serum concentration of 100 nM or even 200 nM,the high subcutaneous bioavailability in combination with the lowclearance facilitates a once weekly subcutaneous dose regimen of 10mg/kg PS10536 in monkey.

Example 15 Production and Characterization of IL-1-1R-I Binding FusionProteins with Mutations in the Fc Portion

Four IL-1-R-I binding fusion proteins of the IL-1R-I binding polypeptidevariant Z18754 (SEQ ID NO:1252) with mutated variants of the Fc portionof IgG1 or IgG4 were produced. In the IgG1 Fc portion, one mutation[N297A] was introduced in order to abolish effector functions throughinteraction with FCγR3A. Fc-containing polypeptides produced inmammalian cells might otherwise cause such effector functions. Inaddition, in one fusion protein (SEQ ID NO:1735) two other residues wereintroduced, denoted LS (WO2009/086320), to increase affinity to FcRn,and in another fusion protein (SEQ ID NO:1736) three mutations denotedYTE (WO02060919) were introduced to increase affinity to FcRn. Onefusion protein of Z18754 (SEQ ID NO:1252) with an IgG4 Fc a subtype,which is known to elicit low effector responses, was produced (SEQ IDNO:1737). One mutation, known as S228P (van der Neut Kolfschoten M. etal., 2007, Science 317(5844):1554-7), was furthermore introduced in thehinge region of this fusion protein. This mutation abolishes the armexchange that may take place for the IgG4 subclass of antibodies.

Material and Methods

The fusion proteins PS10653-PS10656 (SEQ ID NO:1734-1737), see Table 30for specifications, were produced in CHO cells according to Example 10.

The inhibitory effect of IL-1R-I binding fusion proteins on IL-1βinduced IL-6 production in Normal Human Dermal Fibroblasts (NHDF) cellswas monitored as set out in Example 11.

The affinity towards FCγR3A was analysed with Biolayer Interferometryessentially as disclosed in Example 10, with the exception thatbiotinylated FCγR3A (Sino Biological) was immobilized on a Streptavidinbiosensor for kinetics (Pall/Fortebio) and the proteins were testedusing concentrations of 100 and 1000 nM. In addition Human IgG(GammaNorm, Octapharma) was used as control in the measurements.

The affinity towards FcRn was assessed using Biacore essentially asdescribed in Example 9 with the exception that human biotinylated FcRn(Immunitrack ApS) was immobilized on a Biotin CAPture chip (GEHealthcare) and the analytes were tested in five concentrations, rangingfrom 2.5-200 nM.

TABLE 30 Fusion proteins and expression systems SEQ ID Fusion ExpressionNO proteins Construct system 1734 PSI0653 Z18754[N297A]-IgG1 Fc CHO 1735PSI0654 Z18754[N297A]-LS-IgG1 Fc CHO 1736 PSI0655 Z18754[N297A]-YTE-IgG1Fc CHO 1737 PSI0656 Z18754[S228P]-IgG4 Fc CHO

Results

Cultivation and purification: All the IL-1-R-I binding fusion proteinswith mutated Fc portions (Table 30) were expressed at high levels in CHOcells as soluble proteins. Purification resulted in protein preparationswith high purity, which were analyzed by SDS-PAGE stained with CoomassieBlue. The correct identity and molecular weight of each Z variant wereconfirmed by mass spectrometry analysis.

In vitro pharmacological activity: The IL-1β induced IL-6 release fromNHDF cells was reduced in a concentration-dependent manner by theIL-1R-1 binding fusion proteins. The IL-1R-1 binding fusion proteinswere tested one time. The results from this experiment are accounted forin Table 31.

TABLE 31 In vitro IC₅₀ values SEQ ID NO: Fusion proteins IC₅₀ (pM) 1734PSI0653 4.3 1735 PSI0654 2.9 1736 PSI0655 5.9 1737 PSI0656 8.4

FCγR3A affinity analysis: Human IgG bound to FCγR3A with an affinity of57 nM at 1000 nM concentration. The fusion proteins PS10653-PS10655 (SEQID NO 1734-1736) showed no binding at either concentration. PS10656 (SEQID NO 1737) showed a weak binding at 1000 nM but no binding at 100 nM.

Biacore FcRn affinity analysis: The affinity towards FcRn were in theorder SEQ ID NO 1735>SEQ ID NO 1736>SEQ ID NO 1734=SEQ ID NO 1737.

Conclusion: The mutated Fc variants of the fusion proteins (SEQ IDNO:1734:1737) were equal to the previously produced proteins in terms ofquality and activity (see Example 10). The removal of glycan attachmentsite removed the effector function of the IgG1 Fc portion in SEQ IDNO:1734-1736 as binding to FCγR3A was abolished. The IgG4 Fc containingSEQ ID NO:1737 had low affinity towards FCγR3A as expected for an Fc ofthat subclass. The FcRn affinity enhancing mutations in SEQ ID NO:1735and 1736 displayed increased affinity, with SEQ ID NO:1735 showing thegreatest increase.

Itemized Listing of Embodiments

1. IL-1R-I binding polypeptide, comprising an IL-1R-I binding motif BM,which motif consists of an amino acid sequence selected from:

i) (SEQ ID NO: 1686) EX₂X₃X₄X₅X₆X₇EIX₁₀X₁₁LPNLX₁₆RX₁₈QYX₂₁AFIX₂₅X₂₆LX₂₈Dwherein, independently from each other,

-   -   X₂ is selected from A, D, E, F, H, I, L, Q, S, T and V;    -   X₃ is selected from A, D, E, F, H, I, K, L, N, Q, R, S, T, V, W        and Y;    -   X₄ is selected from A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V,        W and Y;    -   X₅ is selected from A, I and V;    -   X₆ is selected from F, H, I, Q, R, T, V and Y;    -   X₇ is selected from A, D, E, F, G, H, I, L, M, Q, S, T, V, W and        Y;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S,        T, V, W and Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K, R and S;    -   X₂₁ is selected from Q, T and V;    -   X₂₅ is selected from I, M, R, V and Y;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F, I, L and M,    -   and

-   ii) an amino acid sequence which has at least 96% identity to the    sequence defined in i) provided that X₅ is I or V.

2. IL-1R-I binding polypeptide, wherein said IL-1R-I binding motifconsists of an amino acid sequence selected from a sequence wherein ini)

-   -   X₂ is selected from A, I, L, T and V;    -   X₃ is selected from E and Y;    -   X₄ is selected from A, E, I, K, Q, R, T, V and Y;    -   X₅ is selected from I and V;    -   X₆ is selected from Q and Y;    -   X₇ is selected from F and M;    -   X₁₀ is selected from F and Y;    -   X₁₁ is selected from A, D, E, F, G, H, K, L, Q, R, S, T, V and        Y;    -   X₁₆ is selected from N and T;    -   X₁₈ is selected from K and R;    -   X₂₁ is selected from T and V;    -   X₂₅ is selected from I and R;    -   X₂₆ is selected from K and S, and    -   X₂₈ is selected from F and L,    -   and an amino acid sequence which has at least 93% identity to        the sequence defined in i).

3. IL-1R-I binding polypeptide according to any one of the precedingitems, wherein sequence i) fulfills at least five of the ten conditionsI-X:

-   -   I. X₃ is E;    -   II. X₅ is selected from I and V;    -   III. X₆ is selected from Q and Y;    -   IV. X₇ is selected from F and M;    -   V. X₁₀ is selected from F and Y;    -   VI. X₁₈ is selected from K and R;    -   VII. X₂₁ is T;    -   VIII. X₂₅ is R;    -   IX. X₂₆ is selected from K and S; and    -   X. X₂₈ is selected from F and L.

4. IL-1R-I binding polypeptide according to item 3, wherein sequence i)fulfills at least six of the ten conditions I-X.

5. IL-1R-I binding polypeptide according to item 4, wherein sequence i)fulfills at least seven of the ten conditions I-X.

6. IL-1R-I binding polypeptide according to item 5, wherein sequence i)fulfills at least eight of the ten conditions I-X.

7. IL-1R-I binding polypeptide according to item 6, wherein sequence i)fulfills at least nine of the ten conditions I-X.

8. IL-1R-I binding polypeptide according to item 7, wherein sequence i)fulfills all of the ten conditions I-X.

9. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₂X₃X₆ is VEQ or VEY.

10. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₂X₃X₆ is IEQ or VEQ.

11. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₆X₁₀ is selected from the group consisting of QF, QY, YF andYY.

12. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₆X₁₀ is QF.

13. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₁₀X₁₈ is selected from the group consisting of FK, FR, YK andYR.

14. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₁₀X₁₈ is FK or FR.

15. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₁₈X₂₅X₂₈ is KRL or RRL.

16. IL-1R-I binding polypeptide according to any one of items 1-8,wherein X₅X₇ is IM or VM.

17. IL-1R-I binding polypeptide according to any preceding item, whereinsequence i) corresponds to the sequence from position 8 to position 36in a sequence selected from the group consisting of SEQ ID NO:1-1632,and 1679, such as the group consisting of SEQ ID NO:20-1632, and 1679.

18. IL-1R-I binding polypeptide according to any preceding item, whereinsequence i) corresponds to the sequence from position 8 to position 36in a sequence selected from the group consisting of SEQ ID NO:1206-1632and 1679, such as the group consisting of SEQ ID NO:1210-1632 and 1679.

19. IL-1R-I binding polypeptide according to any preceding item, whereinsequence i) corresponds to the sequence from position 8 to position 36in a sequence selected from the group consisting of SEQ ID NO:1252,1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1679, such asthe group consisting of SEQ ID NO:1252, 1328, 1435 and 1679.

20. IL-1R-I binding polypeptide according to item 19, wherein sequencei) corresponds to the sequence from position 8 to position 36 in SEQ IDNO:1252.

21. IL-1R-I binding polypeptide according to item 19, wherein sequencei) corresponds to the sequence from position 8 to position 36 in SEQ IDNO:1328.

22. IL-1R-I binding polypeptide according to item 19, wherein sequencei) corresponds to the sequence from position 8 to position 36 in SEQ IDNO:1435.

23. IL-1R-I binding polypeptide according to any preceding item, whereinsaid IL-1R-I binding motif forms part of a three-helix bundle proteindomain.

24. IL-1R-I binding polypeptide according to item 23, wherein saidIL-1R-I binding motif essentially forms part of two helices with aninterconnecting loop, within said three-helix bundle protein domain.

25. IL-1R-I binding polypeptide according to item 24, wherein saidthree-helix bundle protein domain is selected from bacterial receptordomains.

26. IL-1R-I binding polypeptide according to item 25, wherein saidthree-helix bundle protein domain is selected from domains of protein Afrom Staphylococcus aureus or derivatives thereof.

27. IL-1R-I binding polypeptide according to any preceding item, whichcomprises a binding module BMod, the amino acid sequence of which isselected from:

iii) (SEQ ID NO: 1687)K-[BM]-DPSQSX_(a)X_(b)LLX_(c)EAKKLX_(d)X_(e)X_(f)Q;

-   -   wherein    -   [BM] is an IL-1R-I binding motif as defined in any one of items        1-22;    -   X_(a) is selected from A and S;    -   X_(b) is selected from N and E;    -   X_(c) is selected from A, S and C;    -   X_(d) is selected from E, N and S;    -   X_(e) is selected from D, E and S;    -   X_(f) is selected from A and S; and    -   iv) an amino acid sequence which has at least 91% identity to a        sequence defined by iii).

28. IL-1R-I binding polypeptide according to any preceding item, whereinsequence iii) corresponds to the sequence from position 7 to position 55in a sequence selected from the group consisting of SEQ ID NO:1-1638,1667-1668 and 1670-1679; such as the group consisting of SEQ IDNO:20-1638 and 1670-1679.

29. IL-1R-I binding polypeptide according to item 28, wherein sequenceiii) corresponds to the sequence from position 7 to position 55 in asequence selected from the group consisting of SEQ ID NO:1206-1632 and1670-1679, such as the group consisting of SEQ ID NO:1210-1632 and1670-1679.

30. IL-1R-I binding polypeptide according to item 28, wherein sequenceiii) corresponds to the sequence from position 7 to position 55 in asequence selected from the group consisting of SEQ ID NO:1252, 1285,1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679, such asthe group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676 and1679.

31. IL-1R-I binding polypeptide according to item 30, wherein sequenceiii) corresponds to the sequence from position 7 to position 55 in SEQID NO:1672.

32. IL-1R-I binding polypeptide according to item 30, wherein sequenceiii) corresponds to the sequence from position 7 to position 55 in SEQID NO:1675.

33. IL-1R-I binding polypeptide according to item 30, wherein sequenceiii) corresponds to the sequence from position 7 to position 55 in SEQID NO:1676.

34. IL-1R-I binding polypeptide according to any preceding item, whichcomprises an amino acid sequence selected from:

SEQ ID NO: 1695 ADNNFNK-[BM]DPSQSANLLSEAKKLNESQAPK; SEQ ID NO: 1696ADNKFNK-[BM]DPSQSANLLAEAKKLNDAQAPK; SEQ ID NO: 1697ADNKFNK-[BM]DPSVSKEILAEAKKLNDAQAPK; SEQ ID NO: 1698ADAQQNNFNK-[BM]DPSQSTNVLGEAKKLNESQAPK; SEQ ID NO: 1699AQHDE-[BM]DPSQSANVLGEAQKLNDSQAPK; SEQ ID NO: 1700VDNKFNK-[BM]DPSQSANLLAEAKKLNDAQAPK; SEQ ID NO: 1701AEAKYAK-[BM]DPSESSELLSEAKKLNKSQAPK; SEQ ID NO: 1702VDAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1703VDAKYAK-[BM]DPSQSSELLAEAKKLNDSQAPK; SEQ ID NO: 1704AEAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1705AEAKYAK-[BM]DPSQSSELLSEAKKLNDSQAP; SEQ ID NO: 1706AEAKFAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1707AEAKFAK-[BM]DPSQSSELLSEAKKLNDSQAP; SEQ ID NO: 1708AEAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1709AEAKYAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1710AEAKYAK-[BM]DPSQSSELLSEAKKLSESQAP; SEQ ID NO: 1711AEAKFAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1712AEAKFAK-[BM]DPSQSSELLSEAKKLSESQAP; SEQ ID NO: 1713AEAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; SEQ ID NO: 1714AEAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; SEQ ID NO: 1715AEAKYAK-[BM]DPSQSSELLSEAKKLESSQAP; SEQ ID NO: 1716AEAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; SEQ ID NO: 1717AEAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; SEQ ID NO: 1718AEAKYAK-[BM]DPSQSSELLSEAKKLSDSQAP; SEQ ID NO: 1719AEAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; SEQ ID NO: 1720AEAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; SEQ ID NO: 1721VDAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK; SEQ ID NO: 1722VDAKYAK-[BM]DPSQSSELLAEAKKLNDAQAPK; SEQ ID NO: 1723VDAKYAK-[BM]DPSQSSELLSEAKKLSESQAPK; SEQ ID NO: 1724VDAKYAK-[BM]DPSQSSELLAEAKKLSEAQAPK; SEQ ID NO: 1725VDAKYAK-[BM]DPSQSSELLSEAKKLESSQAPK; SEQ ID NO: 1726VDAKYAK-[BM]DPSQSSELLAEAKKLESAQAPK; SEQ ID NO: 1727VDAKYAK-[BM]DPSQSSELLSEAKKLSDSQAPK; SEQ ID NO: 1728VDAKYAK-[BM]DPSQSSELLAEAKKLSDSQAPK; SEQ ID NO: 1729VDAKYAK-[BM]DPSQSSELLAEAKKLSDAQAPK; SEQ ID NO: 1730VDAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK; SEQ ID NO: 1731AEAKYAK-[BM]DPSQSSELLAEAKKLNKAQAPK, and SEQ ID NO: 1732ADAKYAK-[BM]DPSQSSELLSEAKKLNDSQAPK,wherein [BM] is an IL-1R-I binding motif as defined in any one of items1-22.

35. IL-1R-I binding polypeptide according to any one of items 1-34,which comprises an amino acid sequence selected from:

xix) (SEQ ID NO: 1721) VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK

wherein [BM] is an IL-1R-I binding motif as defined in any one of items1-22; and

xx) an amino acid sequence which in the sequences flanking the BM has atleast 89% identity to the sequence defined in xix).

36. IL-1R-I binding polypeptide according to any one of items 1-34,which comprises an amino acid sequence selected from:

xxi) (SEQ ID NO: 1709) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK;

wherein [BM] is an IL-1R-I binding motif as defined in any one of items1-22; and

xxii) an amino acid sequence which in the sequences flanking the BM hasat least 89% identity to the sequence defined in xxi).

37. IL-1R-I binding polypeptide according to item 35 or 36, whereinsequence xix) or xxi) corresponds to the sequence from position 1 toposition 58 in a sequence selected from the group consisting of SEQ IDNO:1-1632, 1667-1668 and 1670-1679; such as the group consisting of SEQID NO:20-1632, 1667-1668 and 1670-1679.

38. IL-1R-I binding polypeptide according to item 37 wherein xix) orxxi) corresponds to the sequence from position 1 to position 58 in asequence selected from the group consisting SEQ ID NO:1206-1632,1667-1668 and 1670-1679, such as the group consisting of SEQ IDNO:1210-1632, 1667-1668 and 1670-1679.

39. IL-1R-I binding polypeptide according to item 38, wherein sequencexix) or xxi) corresponds to the sequence from position 1 to position 58in a sequence selected from the group consisting of SEQ ID NO:1252,1285, 1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679, suchas the group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676and 1679.

40. IL-1R-I binding polypeptide according to item 39, wherein sequencexxi) corresponds to the sequence from position 1 to position 58 in SEQID NO:1672.

41. IL-1R-I binding polypeptide according to item 39, wherein sequencexxi) corresponds to the sequence from position 1 to position 58 in SEQID NO:1675.

42. IL-1R-I binding polypeptide according to item 39, wherein sequencexxi) corresponds to the sequence from position 1 to position 58 in SEQID NO:1676.

43. IL-1R-I binding polypeptide according to any preceding item, whichis capable of blocking IL-1R-I dependent signaling.

44. IL-1R-I binding polypeptide according to item 43, wherein the halfmaximal inhibitory concentration (IC₅₀) of the blocking is at most1×10⁻⁷ M, such as at most, 1×10⁻⁸ M, such as at most 1×10⁻⁹ M, such asat most 5×10⁻¹⁰ M.

45. IL-1R-I binding polypeptide according to item 43 or 44, which iscapable of blocking the interaction of IL-1R-I with IL-1 cytokines, suchas the interaction of IL-1R-I with IL-1α and/or IL-1β.

46. IL-1R-I binding polypeptide according to any preceding item which iscapable of binding to IL-1R-I such that the EC₅₀ value of theinteraction is at most 1×10⁻⁷ M, such as at most 1×10⁻⁸ M, such as atmost 5×10⁻⁹ M, such as at most 1×10⁻¹⁰ M such as at most 5×10⁻¹⁰ M, suchas at most 2×10⁻¹⁰ M.

47. IL-1R-I binding polypeptide according to any preceding item which iscapable of binding to IL-1R-I such that the K_(D) value of theinteraction is at most 1×10⁻⁶ M, such as at most 1×10⁻⁷ M, such as atmost 1×10⁻⁸ M, such as at most 1×10⁻⁹ M, such as at most 9×10⁻¹⁰.

48. IL-1R-I binding polypeptide according to any one of items 43-47,wherein said IL-1R-I is human IL-1R-I or cynomolgus IL-1R-I, such ashuman IL-1R-I.

49. IL-1R-I binding polypeptide according to any preceding item whichcomprises additional amino acids at the C-terminal and/or N-terminalend.

50. IL-1R-I binding polypeptide according to item 49, wherein saidadditional amino acid(s) improve(s) production, purification,stabilization in vivo or in vitro, coupling or detection of thepolypeptide.

51. IL-1R-I binding polypeptide according to any preceding item inmultimeric form, comprising at least two IL-1R-I binding polypeptidemonomer units, the amino acid sequences of which are the same ordifferent.

52. IL-1R-I binding polypeptide according to item 51, wherein saidIL-1R-I binding polypeptide monomer units are covalently coupledtogether.

53. IL-1R-I binding polypeptide according to item 52, wherein theIL-1R-I binding polypeptide monomer units are expressed as a fusionprotein.

54. IL-1R-I binding polypeptide according to any one of items 51-53, indimeric form.

55. Fusion protein or conjugate comprising

-   -   a first moiety consisting of an IL-1R-I binding polypeptide        according to any preceding item; and    -   a second moiety consisting of a polypeptide having a desired        biological activity.

56. Fusion protein or conjugate according to item 55, wherein saiddesired biological activity is a therapeutic activity.

57. Fusion protein or conjugate according to item 55, wherein saiddesired biological activity is a binding activity.

58. Fusion protein or conjugate according to item 55, wherein saiddesired biological activity is a binding activity modifying tissuedistribution of said fusion protein or conjugate.

59. Fusion protein or conjugate according to item 57, wherein saiddesired biological activity is an in vivo half-life increasing activitysuch that said second moiety increases in vivo half-life of the fusionprotein or conjugate.

60. Fusion protein or conjugate according to item 59, said in vivohalf-life increasing activity is an albumin binding activity.

61. Fusion protein or conjugate according to item 60, wherein saidalbumin binding activity is provided by the albumin binding domain ofstreptococcal protein G or a derivative thereof.

62. Fusion protein or conjugate according to item 61, wherein saidsecond moiety comprises a polypeptide having an amino acid sequence asset out in SEQ ID NO:1659-1661, such as SEQ ID NO:1661.

63. Fusion protein or conjugate according to item 59, wherein saidsecond moiety is serum albumin.

64. Fusion protein or conjugate according to item 59, wherein saidsecond moiety is an Fc portion of an antibody, such as an IgG1 Fc or anIgG4 Fc.

65. Fusion protein or conjugate according to item 64, wherein said Fc isa mutated Fc which relative to an unmutated form of Fc displays improvedaffinity to FcRn and/or reduced effector response.

66. Fusion protein or conjugate according to item 58 or 59, wherein saidsecond moiety is transferrin.

67. Fusion protein or conjugate according to any one of items 59-65,comprising a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1639-1658 and 1734-1737, such as from thegroup consisting of SEQ ID NO:1646-1654 and 1734-1737.

68. Fusion protein or conjugate according to item 57, wherein saidbinding activity acts to block a biological activity.

69. Fusion protein or conjugate according to any one of items 55-68,further comprising at least one linker, said linker optionally beingselected from the group consisting of flexible amino acid linkers, rigidamino acid linkers and cleavable amino acid linkers.

70. Fusion protein or conjugate according item 69, wherein said linkeris arranged between said first moiety and said second moiety.

71. Fusion protein or conjugate according item 69, wherein said linkeris arranged within said first moiety.

72. Fusion protein or conjugate according item 69 or 70, wherein saidlinker is a flexible linker comprising at least one amino acidresidue(s) selected from the group consisting of glycine, serine andalanine.

73. Fusion protein or conjugate according to item 72, wherein saidlinker is selected from the group consisting of GS, VDSS, VDGS, VEGS,ASGS, (GGGGS)₂ (SEQ ID NO:1683), AS(GGGGS)₂ (SEQ ID NO:1684) and((KEAAA)₃KELAA)₂ (SEQ ID NO:1685).

74. Fusion protein or conjugate according to item 73, wherein saidlinker is selected from a AS(GGGGS)₂ (SEQ ID NO:1684) and((KEAAA)₃KELAA)₂ (SEQ ID NO:1685).

75. A polynucleotide encoding a polypeptide or a fusion proteinaccording to any one of items 1-74.

76. Expression vector comprising a polynucleotide according to item 75.

77. Host cell comprising an expression vector according to item 76.

78. Method of producing a polypeptide according to any one of items1-77, comprising

-   -   culturing a host cell according to item 77 under conditions        permissive of expression of said polypeptide from said        expression vector, and    -   isolating said polypeptide.

79. Composition comprising an IL-1R-I binding polypeptide, fusionprotein, or conjugate according to any one of items 1-74 and at leastone pharmaceutically acceptable excipient or carrier.

80. Composition according to item 79, further comprising at least oneadditional active agent, such as an agent selected from an immuneresponse modifying agent and an anti-cancer agent.

81. Composition according to item 79 or 80, wherein said fusion proteinis a fusion protein according to any one of claims 59-67.

82. IL-1R-I binding polypeptide, fusion protein, or conjugate accordingto any one of items 1-74 or a composition according to any one of items79-81 for use as a medicament, a diagnostic agent and/or a prognosticagent.

83. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition according to item 82 for use as a medicament.

84. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition for use according to item 83, wherein said polypeptide,fusion protein, conjugate or composition modulates IL-1R-I function invivo.

85. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition according to item 82 for use as a diagnostic agent and/or aprognostic agent.

86. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition for use according to any one of items 82-85, in thetreatment, prognosis or diagnosis of an IL-1R-I related disorder.

87. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition for use according to item 86, wherein said IL-1R-I relateddisorder is selected from the group consisting of inflammatory disease,auto-inflammatory syndromes, autoimmune disease, infectious disease,cardiovascular disease, ischaemic disease, cancer and diabetes.

88. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition for use according to item 86 or 87, wherein said IL-1R-Irelated disorder is selected from the group consisting of familialMediterranean fever (FMF); cryopyrin-associated periodic syndrome(CAPS); TNF receptor-associated periodic syndrome (TRAPS); hyper-IgDsyndrome (HIDS); periodic fever; aphthous stomatitis; pharyngitis;adenitis (PFAPA); rheumatoid arthritis (RA), juvenile RA, juvenileidiopathic arthritis, systemic juvenile idiopathic arthritis;adult-onset Still's disease; Schnitzler syndrome; Muckle Wells Syndrome;macrophage activation syndrome; Behget's disease; uveitis; acnevulgaris; pyoderma gangrenosum; gout; type 2 diabetes, new-onsetdiabetes; dry eye syndrome; hidradenitis suppurativa; neutrophilicdermatoses, in particular cytophagic histiocytic panniculitis,Weber-Christian disease, and neutrophilic panniculitis; cardiovasculardisease, myocardial infarction, stroke; liver failure, kidney failure;acute lung injury; pseudogout, calcium-pyrophosphate depositiondisorder, chondrocalcinosis, deficiency of the IL-1 receptor antagonist(DIRA), deficiency of the IL-36 receptor antagonist (DITRA), ADAM2deficiency (DADA2), pyogenic arthritis, pyoderma gangrenosum and acne(PAPA) syndrome, pyoderma gangrenosum, acne and suppurative hidradenitis(PASH) syndrome, PAPA and suppurative hidradenitis (PAPASH) syndrome,autoinflammatory syndrome with lymphedema (AISLE), PhospholipaseC-gamma-2 mutation autoinflammatory syndrome, NALP12-associated periodicsyndrome (NAPS12), mevalonate kinase deficiency (MKD), psoriaticarthritis, reactive arthritis, ankylosing spondylitis,haemochromatosis-related arthritis, periarticular calcinosis,osteoarthritis, inflammatory osteoarthritis, hand osteoarthritis,pustular psoriasis such as generalised pustular psoriasis (GPP),palmoplantar pustulosis (PPP), acrodermatitis continua of Hallopeau(ACH); Blau syndrome; Sweet syndrome; various vasculitides such asgiant-cell arteritis (GCA), polymyalgia rheumatica (PMR), Takayasuarteritis, Kawasaki disease, urticarial vasculitis, and Henoch-Schönleinpurpura (HSP); neutrophilic urticaria and idiopathic cold urticaria;lichen planus; polymyositis, dermatomyositis, juvenile dermatomyositisand inclusion-body myositis; various stages of myeloma such assmoldering myeloma (SMM), indolent myeloma, Waldenstrom'smacroglobulinemia, and multiple myeloma; tumor-induced cachexia; solidtumor growth; neonatal disorders such as bronchopulmonary dysplasia(prophylaxis), necrotising enterocolitis (NEC), retinopathy ofprematurity (ROP), cerebral palsy due to perinatal cerebral ischemia,and infant respiratory distress syndrome (IRDS); Whipple's disease;traumatic brain injury; refractory epilepsy; systemic inflammatoryresponse syndrome (SIRS); cutaneous lupus; Jessner-Kanof disease;amyotrophic lateral sclerosis; systemic sclerosis (scleroderma); septicshock; acute pancreatitis; chronic recurrent multifocal osteomyelitis,non-bacterial osteitis (NBO), synovitis, acne, pustulosis, hyperostosis,osteitis (SAPHO) syndrome, and Majeed syndrome; relapsingpolychondritis; idiopathic recurrent pericarditis (IRP); myocarditis;Erdheim-Chester disease; juvenile xantogranuloma; islet-celltransplantation; haemodialysis-induced systemic inflammation;graft-versus-host disease; ANCA-associated glomerulonephritis andrecurrent glomerulonephritis; Cogan syndrome; autoimmune inner-eardisease; chronic granulomatous disease (CGD); Castleman's disease;cardiac failure; diastolic cardiac failure; antisynthetase syndrome;acute ACL injury; acute haemorrhagic leukoencephalitis; AA amyloidosis;Di George syndrome; generalised fatigue; chronic fatigue syndrome (CFS);gulf-war illness (GWI); and narcolepsy.

89. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition for use according to item 87, wherein said IL-1R-I relateddisorder is cancer, such as a cancer selected from the group consistingof multiple myeloma, colon cancer, breast cancer, lung cancer, head andneck cancer, melanoma and prostate cancer.

90. IL-1R-I binding polypeptide, fusion protein, conjugate orcomposition for use according to any one of items 82-89, wherein saidIL-1R-I binding polypeptide, fusion protein, conjugate or composition isadministered repeatedly within a 24 hour period from disease onset, suchas at least two times within a 24 hour period from disease onset, suchas at least three times within a 24 hour period from disease onset, suchas continuously during 24 hours from disease onset, to a subject in needthereof.

91. Fusion protein, conjugate or composition for use according to anyone of items 82-84, 86-89, wherein said fusion protein or conjugate is afusion protein or conjugate according to any one of items 58-67 and saidcomposition is a composition according to item 81.

92. Fusion protein, conjugate or composition for use according to item

91, wherein said fusion protein, conjugate or composition isadministered once weekly to a subject in need thereof.

93. Method of treatment of an IL-1R-I related disorder, comprisingadministering to a subject in need thereof an effective amount of anIL-1R-I binding polypeptide, fusion protein or conjugate according toany one of items 1-74 or a composition according to any one of items79-81.

94. Method according to item 93, wherein said IL-1R-I related disorderis selected from the group consisting inflammatory disease,auto-inflammatory syndrome, autoimmune disease, infectious disease,cardiovascular disease, ischaemic disease, cancer and diabetes.

95. Method according to item 93 or 94, wherein said IL-1R-I relateddisorder is selected from the group as defined in item 88.

96. Method according to item 94, wherein said IL-1R-I related disorderis cancer, such as a cancer selected from the group consisting ofselected from the group consisting of multiple myeloma, colon cancer,breast cancer, lung cancer, head and neck cancer, melanoma and prostatecancer.

97. Method according to any one of items 93-96, wherein said IL-1R-Ibinding polypeptide, fusion protein or conjugate or composition isadministered repeatedly within a 24 h period from disease onset, such asat least two times within a 24 hour period from disease onset, such asat least three times within a 24 hour period from disease onset, such ascontinuously during 24 hours from disease onset, to a subject in needthereof.

98. Method according to any one of claims 93-96, wherein said fusionprotein or conjugate is a fusion protein or conjugate according to anyone of items 59-67 and said composition is a composition according toitem 81.

99. Method according to item 97, wherein said fusion protein, conjugateor composition is administered once weekly to a subject in need thereof.

1. IL-1R-1 binding polypeptide, comprising an IL-1R-1 binding motif BM,which motif consists of an amino acid sequence selected from: i)(SEQ ID NO: 1686) EX₂X₃X₄X₅X₆X₇EIX₁₀X₁₁LPNLX₁₆RX₁₈QYX₂₁AFIX₂₅X₂₆LX₂₈D

wherein, independently from each other, X₂ is selected from A, I, L, Tand V; X₃ is selected from E and Y; X₄ is selected from A, E, I, K, Q,R, T, V and Y; X₅ is selected from I and V; X₆ is selected from Q and Y;X₇ is selected from F and M; X₁₀ is selected from F and Y; X₁₁ isselected from A, D, E, F, G, H, K, L, Q, R, S, T, V and Y; X₁₆ isselected from N and T; X₁₈ is selected from K and R; X₂₁ is selectedfrom T and V; X₂₅ is selected from I and R; X₂₆ is selected from K andS, and X₂₈ is selected from F and L, and ii) an amino acid sequencewhich has at least 93% identity to the sequence defined in i) providedthat X₅ is I or V.
 2. (canceled)
 3. IL-1R-1 binding polypeptideaccording to claim 1, wherein sequence i) fulfills at least five, suchas at least six, at least seven, at least eight, at least nine or all ofthe ten conditions I-X: I. X₃ is E; II. X₅ is selected from I and V;III. X₆ is selected from Q and Y; IV. X₇ is selected from F and M; V.X₁₀ is selected from F and Y; VI. X₁₈ is selected from K and R; VII. X₂₁is T; VIII. X₂₅ is R; IX. X₂₆ is selected from K and S; and X. X₂₈ isselected from F and L. 4.-6. (canceled)
 7. IL-1R-1 binding polypeptideaccording to claim 1, wherein said IL-1R-1 binding motif forms part of athree-helix bundle protein domain, wherein said IL-1R-1 binding motifpreferably essentially forms part of two helices with an interconnectingloop, within said three-helix bundle protein domain.
 8. (canceled) 9.IL-1R-1 binding polypeptide according to claim 1, which comprises anamino acid sequence selected from: xix)VDAKYAK-[BM]-DPSQSSELLSEAKKLNDSQAPK (SEQ ID NO:1721) wherein [BM] is anIL-1R-1 binding motif as defined in claim 1; xx) an amino acid sequencewhich in the sequences flanking the BM has at least 89% identity to thesequence defined in xix); xxi) AEAKYAK-[BM]-DPSQSSELLSEAKKLSESQAPK (SEQID NO:1709) wherein [BM] is an IL-1R-1 binding motif as defined in claim1; and xxii) an amino acid sequence which in the sequences flanking theBM has at least 89% identity to the sequence defined in xxi). 10.(canceled)
 11. IL-1R-1 binding polypeptide according to claim 9, whereinsequence xix) or xxi) corresponds to the sequence from position 1 toposition 58 in a sequence selected from the group consisting of SEQ IDNO:1-1632, 1667-1668 and 1670-1679; such as the group consisting of SEQID NO:20-1632, 1667-1668 and 1670-1679.
 12. IL-1R-1 binding polypeptideaccording to claim 9 wherein xix) or xxi) corresponds to the sequencefrom position 1 to position 58 in a sequence selected from the groupconsisting SEQ ID NO:1206-1632, 1667-1668 and 1670-1679, such as thegroup consisting of SEQ ID NO:1210-1632, 1667-1668 and 1670-1679. 13.IL-1R-1 binding polypeptide according to claim 9, wherein sequence xix)or xxi) corresponds to the sequence from position 1 to position 58 in asequence selected from the group consisting of SEQ ID NO:1252, 1285,1307, 1308, 1328, 1331, 1415, 1421, 1435, 1594 and 1670-1679, such asthe group consisting of SEQ ID NO:1252, 1328, 1435, 1672, 1675-1676 and1679.
 14. IL-1R-1 binding polypeptide according to claim 1, which iscapable of blocking the interaction of IL-1R-1 with IL-1 cytokines, suchas the interaction of IL-1R-1 with IL-1α and/or IL-1β.
 15. IL-1R-1binding polypeptide according to claim 1, which is capable of binding toIL-1R-1 such that the K_(D) value of the interaction is at most 1×10⁻⁶M, such as at most 1×10⁻⁷ M, such as at most 1×10⁻⁸ M, such as at most1×10⁻⁹ M, such as at most 9×10⁻¹⁰. 16.-17. (canceled)
 18. Fusion proteinor conjugate comprising a first moiety consisting of an IL-1R-1 bindingpolypeptide according to claim 1; and a second moiety consisting of apolypeptide having a desired biological activity. 19.-21. (canceled) 22.Fusion protein or conjugate according to claim 18, wherein said desiredbiological activity is an in vivo half-life increasing activity suchthat said second moiety increases in vivo half-life of the fusionprotein or conjugate.
 23. Fusion protein or conjugate according to claim18, wherein said second moiety is selected from the group consisting ofthe albumin binding domain of streptococcal protein G or a derivativethereof; serum albumin; an Fc portion of an antibody, and transferrin.24. Fusion protein or conjugate according to claim 23, wherein saidsecond moiety is an Fc portion of an antibody, such as an IgG1 Fc or anIgG4 Fc.
 25. (canceled)
 26. Fusion protein or conjugate according toclaim 18, comprising a polypeptide having an amino acid sequenceselected from the group consisting of SEQ ID NO:1639-1658 and 1734-1737,such as from the group consisting of SEQ ID NO:1648-1654 and 1734-1737.27.-29. (canceled)
 30. Composition comprising an IL-1R-1 bindingpolypeptide, fusion protein, or conjugate according to claim 1 and atleast one pharmaceutically acceptable excipient or carrier. 31.-39.(canceled)
 40. IL-1R-1 binding polypeptide, comprising an IL-1R-1binding motif BM, which motif consists of an amino acid sequenceselected from: i) a sequence from position 8 to position 36 in asequence selected from the group consisting of SEQ ID NO:1-1632, and1679, such as the group consisting of SEQ ID NO:20-1632 and 1679, andii) an amino acid sequence which has at least 96% identity to thesequence defined in i) provided that X₅ is I or V.
 41. IL-1R-1 bindingpolypeptide according to claim 40, wherein sequence i) corresponds tothe sequence from position 8 to position 36 in a sequence selected fromthe group consisting of SEQ ID NO:1206-1632 and 1679, such as the groupconsisting of SEQ ID NO:1210-1632 and
 1679. 42. IL-1R-1 bindingpolypeptide according to claim 40, wherein sequence i) corresponds tothe sequence from position 8 to position 36 in a sequence selected fromthe group consisting of SEQ ID NO:1252, 1285, 1307, 1308, 1328, 1331,1415, 1421, 1435, 1594 and 1679, such as the group consisting of SEQ IDNO:1252, 1328, 1435 and
 1679. 43. Method of treatment of an IL-1R-1related disorder, comprising administering to a subject in need thereofan effective amount of an IL-1R-1 binding polypeptide according toclaim
 1. 44. Method of treatment of an IL-1R-1 related disorder,comprising administering to a subject in need thereof an effectiveamount of an IL-1R-1 binding polypeptide according to claim 1, whereinsaid IL-1R-1 related disorder is selected from the group consisting ofinflammatory disease, auto-inflammatory syndromes, autoimmune disease,infectious disease, cardiovascular disease, ischaemic disease, cancerand diabetes.